ErbB surface receptor complexes as biomarkers

ABSTRACT

The invention is directed to a new class of biomarker in patient samples comprising dimers of ErbB cell surface membrane receptors. In one aspect, the invention includes a method of determining the status of a disease or healthful condition by correlating such condition to amounts of one or more dimers of ErbB cell surface membrane receptors measured directly in a patient sample, in particular a fixed tissue sample. In another aspect, the invention includes a method of determining a status of a cancer in a specimen from an individual by correlating measurements of amounts of one or more dimers of ErbB cell surface membrane receptors in cells of the specimen to such status, including presence or absence of a pre-cancerous state, presence or absence of a cancerous state, prognosis of a cancer, or responsiveness to treatment. Preferably, methods of the invention are implemented by using sets of binding compounds having releasable molecular tags that are specific for multiple components of one or more types of receptor dimers. After binding, molecular tags are released and separated from the assay mixture for analysis.

[0001] This is a continuation-in-part of U.S. patent application Ser.No. 10/623,057 filed 17 Jul. 2003; priority is further claimed underU.S. provisional applications Ser. No. 60/459,888 filed 1 Apr. 2003;Ser. No. 60/494,482 filed 11 Aug. 2003; Ser. No. 60/508,034 filed 1 Oct.2003; Ser. No. 60/512,941 filed 20 Oct. 2003; and Ser. No. 60/523,258filed 18 Nov. 2003, all of the above of which are incorporated in theirentirety by reference.

FIELD OF THE INVENTION

[0002] The present invention relates generally to biomarkers, and moreparticularly, to the use of ErbB cell surface receptor complexes, suchas dimers and oligomers, as biomarkers.

BACKGROUND OF THE INVENTION

[0003] A biomarker is a characteristic that is objectively measured andevaluated as an indicator of normal biological processes, pathogenicprocesses, or pharmacological responses to a therapeutic intervention,Atkinson et al, Clin. Pharmacol. Ther., 69: 89-95 (2001). Biomarkersvary widely in nature, ease of measurement, and correlation withphysiological states of interest, e.g. Frank et al, Nature Reviews DrugDiscovery, 2: 566-580 (2003). It is widely believed that the developmentof new validated biomarkers will lead both to significant reductions inhealthcare and drug development costs and to significant improvements intreatment for a wide variety of diseases and conditions. Thus, a greatdeal of effort has been directed to using new technologies to find newclasses of biomarkers, e.g. Petricoin et al, Nature Reviews DrugDiscovery, 1: 683-695 (2002); Sidransky, Nature Reviews Cancer, 2:210-219 (2002).

[0004] The interactions of cell surface membrane components play crucialroles in transmitting extracellular signals to a cell in normalphysiology, and in disease conditions. In particular, many types of cellsurface receptors undergo dimerization, oligomerization, or clusteringin connection with the transduction of an extracellular event or signal,e.g. ligand-receptor binding, into a cellular response, such asproliferation, increased or decreased gene expression, or the like, e.g.George et al, Nature Reviews Drug Discovery, 1: 808-820 (2002); Melladoet al, Ann. Rev. Immunol., 19: 397-421 (2001); Schlessinger, Cell, 103:211-225 (2000); Yarden, Eur. J. Cancer, 37: S3-S8 (2001). The role ofsuch signal transduction events in diseases, such as cancer, has beenthe object of intense research and has led to the development of severalnew drugs and drug candidates, e.g. Herbst and Shin, Cancer, 94:1593-1611 (2002); Yarden and Sliwkowski, Nature Reviews Molecular CellBiology, 2: 127-137 (2001); McCormick, Trends in Cell Biology, 9: 53-56(1999); Blume-Jensen and Hunter, Nature, 411: 355-365 (2001).

[0005] Expression levels of individual cell surface receptors have beenused successfully as biomarkers, e.g. Slamon et al, U.S. Pat. No.4,968,603 (Her2 expression). However, individual receptor expressionlevel alone is not always a reliable indicator of a disease status orcondition, e.g. Chow et al, Clin. Cancer Res., 7: 1957-1962 (2001)(EGFR, or Her1, expression). Despite the important role that receptordimerization plays in cellular and disease processes, receptor dimerexpression has not been employed as a biomarker, in part due to theinconvenience and lack of sensitivity of current measurementtechnologies and the inability or impracticality of using suchtechnologies to carry out measurements on patient samples, which may beformalin fixed and/or in too small a quantity for analysis, e.g. Priceet al, Methods in Molecular Biology, 218: 255-267 (2003); Stagljar,Science STKE 2003, pe56 (2003); Koll et al, International patentpublication WO 2004/008099; Golemis, editor, Protein-ProteinInteractions (Cold Spring Harbor Laboratory Press, New York, 2002);Sorkin et al, Curr. Biol., 10: 1395-1398 (2000); McVey et al, J. Biol.Chem., 17: 14092-14099 (2001); Salim et al, J. Biol. Chem., 277:15482-15485 (2002); Angers et al, Annu. Rev. Pharmacol. Toxicol., 42:409-435 (2002); Szollosi et al, Reviews in Molecular Biotechnology, 82:251-266 (2002); Matko et al, Meth. in Enzymol., 278: 444-462 (1997);Reed-Gitomer, U.S. Pat. No. 5,192,660.

[0006] In view of the above, the availability of a new class ofbiomarkers in patient samples based on the presence, absence, and/orprofile or ratios of cell surface receptor dimers or complexes involvedwith key intracellular processes, such as signal transduction, wouldadvance the field of medicine by providing a new tool for diagnosis,prognosis, patient stratification, and drug development.

SUMMARY OF THE INVENTION

[0007] The invention is directed to biomarkers comprising ErbB receptorcomplexes in cell surface membranes of patient cell or tissue samples,particularly samples preserved by conventional procedures, such asfreezing or fixation. In one aspect, the invention includes a method ofdetermining the status of a disease or healthful condition bycorrelating such condition to amounts of one or more ErbB receptorcomplexes in cell surface membranes in a cell or tissue sample from anindividual. In another aspect, the invention includes a method ofdetermining a status of a cancer in a specimen from an individual bycorrelating measurements of amounts of one or more ErbB surface receptorcomplexes in the specimen to such status. The invention additionallyprovides a method of predicting the effectiveness of ErbB-dimer-actingdrugs, for example, in cancer therapy, by relating numbers and types ofdrug-responsive ErbB dimers to efficacy, or a likelihood of patientresponsiveness.

[0008] In one aspect, the invention permits the determination of adisease status of a patient suffering from a disease characterized byaberrant expression of one or more ErbB cell surface receptor complexesby the following steps: (i) measuring an amount of each of one or moreErbB cell surface receptor complexes in a patient sample; (ii) comparingeach such amount to its corresponding amount in a reference sample; and(iii) correlating differences in the amounts from the patient sample andthe respective corresponding amounts from the reference sample to thedisease status the patient. A patient sample may be fixed or frozen;however, preferably, a patient sample is fixed using conventionalprotocols.

[0009] In a particular aspect, the invention provides a method ofdetermining from measurements on patient samples, especially fixedsamples, the disease status of a patient suffering from a cancer,wherein such measurement are of the types and/or amounts of ErbBreceptor complexes, which are also referred to herein as “Her receptorcomplexes.” Such receptor complexes include, but are not limited to, oneor more of Her1-Her1 homodimers, Her2-Her2 homodimers, Her1-Her2receptor dimers, Her2-Her3 receptor dimers, Her1-Her3 receptor dimers,Her2-Her4 receptor dimers, Her1-PI3K complexes, Her2-PI3K complexes,Her3-PI3K complexes, Her1-SHC complexes, Her2-SHC complexes, Her3-SHCcomplexes, Her1-IGF-1R receptor dimers, Her2-IGF-1R receptor dimers,Her3-IGF-1R receptor dimers, Her1-PDGFR receptor dimers, Her2-PDGFRreceptor dimers, Her3-PDGFR receptor dimers, p95Her2-Her3 receptordimers, p95Her2-Her2 receptor dimers, p95Her2-Her1 receptor dimers,EGFRvIII-Her1 receptor dimers, EGFRvIII-Her2 receptor dimers, andEGFRvIII-Her3 receptor dimers. In other embodiments, such Her receptorcomplexes are selected from the group consisting of Her1-Her2 receptordimers and Her2-Her3 receptor dimers; or the group consisting ofHer1-Her2 receptor dimers, Her2-Her3 receptor dimers, and Her1-Her3receptor dimers. In another embodiment, the invention includesmeasurement of complexes comprising a Her receptor and an intracellularadaptor molecule, particularly, intracellular adaptor molecules thatform complexes with a Her receptor in response to phosphorylation ofsuch receptor. Exemplary receptor complexes of Her receptors andintracellular adaptor molecules include complexes selected from thegroup consisting of Her1-PI3K complexes, Her2-PI3K complexes, Her3-PI3Kcomplexes, Her1-SHC complexes, Her2-SHC complexes, and Her3-SHCcomplexes. The invention further includes the association of receptorheterodimers comprising a Her receptor and another receptor tyrosinekinase to a disease status. Exemplary receptor complexes of Herreceptors and other receptor tyrosine kinases include receptor complexesselected from the group consisting of Her1-IGF-1R receptor dimers,Her2-IGF-1R receptor dimers, Her3-IGF-1R receptor dimers, Her1-PDGFRreceptor dimers, Her2-PDGFR receptor dimers, and Her3-PDGFR receptordimers. The invention further includes the association of receptordimers comprising a full-length Her receptor and a truncated Herreceptor to a disease status. Exemplary receptor complexes offull-length Her receptors and truncated Her receptors include receptorcomplexes selected from group consisting of p95Her2-Her3 receptordimers, EGFRvIII-Her1 receptor dimers, EGFRvIII-Her2 receptor dimers,and EGFRvIII-Her3 receptor dimers. In another aspect, such method ofdetermining disease status includes determining the effectiveness of, orthe responsiveness of a patient to, dimer-acting drugs for treatingcancer, the dimer-acting drug acting on Her receptor complexes asdescribed above.

[0010] In another aspect the invention includes improved determinationsof a disease status by measuring expression of Her1-Her3 receptorcomplexes in a patient sample, as well as expression of Her1-Her2 andHer2-Her3 receptor complexes.

[0011] In another aspect, the invention provides a method of determininga status of a cancer in a patient by determining amounts of one or moredimers of ErbB cell surface membrane receptors or relative amounts of aplurality of dimers of cell surface membrane receptors in a cell ortissue sample from such patient. In one embodiment, such dimers aremeasured using at least two reagents, referred to herein as reagentpairs, that are specific for different members of each dimer: onereagent, referred to herein as a cleaving probe, has a cleavage-inducingmoiety that may be induced to cleave susceptible bonds within itsimmediate proximity; and the other reagent, referred to herein as abinding compound, has one or more molecular tags attach by linkages thatare cleavable by the cleavage-inducing moiety. In accordance with theembodiment, whenever such different members form a dimer, the cleavablelinkages are brought within the effective cleaving proximity of thecleavage-inducing moiety so that molecular tags are released. Thereleased molecular tags are then separated from the reaction mixture andquantified to provide a measure of dimer formation.

[0012] In another aspect of the invention, ErbB receptor dimers in apatient sample are measured ratiometrically; that is, the amount of anErbB dimer is given as a ratio of a measure of one component present inthe dimer to a measure of the total amount of the other component of thedimer, whether it is present in the dimer or in monomeric form. In oneembodiment, typical measures include peak height or peak area of peaksin an electropherogram that are correlated to particular molecular tags.

[0013] In a particular embodiment of this aspect, the invention providesa method of determining a status of a cancer in a patient bysimultaneously determining amounts of a plurality of Her receptor dimersin a fixed tissue sample from the patient. Such dimers may be measuredusing at least two reagents that are specific for different members ofeach dimer: one reagent, referred to herein as a cleaving probe, has acleavage-inducing moiety that may be induced to cleave susceptible bondswithin its immediate proximity; and the other reagent, referred toherein as a binding compound, has one or more molecular tags attach bylinkages that are cleavable by the cleavage-inducing moiety. Inaccordance with the embodiment, whenever Her receptor dimers form, thecleavable linkages of the binding compounds are brought within theeffective cleaving proximity of the cleavage-inducing moiety so thatmolecular tags are released. The molecular tags are then separated fromthe reaction mixture and quantified to provide a measure of Her receptordimer populations. In another embodiment of this aspect, relativeamounts of a plurality of Her receptor dimers are measured and relatedto a status of a cancer in a patient. Exemplary cancers include, but arenot limited to, breast cancer, ovarian cancer, and prostate cancer.Exemplary Her receptor dimers include, but are not limited to, Her1-Her2receptor dimers, Her1-Her3 receptor dimers, Her2-Her3 receptor dimers,and Her2-Her4 receptor dimers, as well as those listed above.

[0014] The present invention provides biomarkers comprising measures ofthe amounts of ErbB receptor complexes in patient samples. Inparticular, profiles of ErbB receptor complex populations may becorrelated to disease status of a patient, and in some embodiments, toprognosis, efficacy of ErbB dimer-acting drugs, and likelihood ofpatient responsiveness to therapy. In accordance with the invention,short comings in the art are overcome by enabling the direct measurementof ErbB receptor complexes in patient samples without the need toculture or otherwise process the cell or tissue samples bymethodologies, such as xenografting, that increase cost and labor aswell as introducing sources of noise and potential artifacts into thefinal assay readouts. The present invention also provides a surrogatemeasurement for intracellular receptor phosphorylation, or othermodifications that are easily destroyed in sample preparationprocedures. Such surrogate measurements are based on the measurement ofcomplexes, such as PI3K or SHC-receptor complexes, and the like, thatdepend on the above modifications for their formation and that are lessaffected by sample preparation procedures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIGS. 1A-1F illustrate diagrammatically the use of releasablemolecular tags to measure receptor dimer populations.

[0016]FIGS. 1G-1H illustrate diagrammatically the use of releasablemolecular tags to measure cell surface receptor complexes in fixedtissue specimens.

[0017]FIGS. 2A-2E illustrate diagrammatically an embodiment of themethod of the invention for profiling relative amounts of dimers of aplurality of receptor types.

[0018]FIGS. 3A-3D illustrate diagrammatically methods for attachingmolecular tags to antibodies.

[0019]FIGS. 4A-4E illustrate data from assays on SKBR-3 and BT-20 celllysates for receptor heterodimers using a method of the invention.

[0020]FIGS. 5A-5C illustrate data from assays for receptor heterodimerson human normal and tumor breast tissue samples using a method of theinvention.

[0021]FIGS. 6A and 6B illustrate data from assays of the invention fordetecting homodimers and phosphorylation of Her1 in lysates of BT-20cells.

[0022]FIG. 7 shows data from assays of the invention that show Her2homodimer populations on MCF-7 and SKBR-3 cell lines.

[0023]FIGS. 8A-8B show data from assays of the invention that detectheterodimers of Her1 and Her3 on cells in response to increasingconcentrations of heregulin (HRG).

[0024]FIGS. 9A and 9B show data on the increases in the numbers ofHer1-Her3 heterodimers on 22Rv1 and A549 cells, respectively, withincreasing concentrations of epidermal growth factor (EGF).

[0025]FIGS. 10A-10C show data on the expression of heterodimers of IGF-1R and various Her receptors in frozen samples from human breast tissue.

[0026]FIGS. 11A-11D illustrate the assay design and experimental resultsfor detecting a PI3 kinase-Her3 receptor activation complex.

[0027]FIGS. 12A-12D illustrate the assay design and experimental resultsfor detecting a Shc/Her3 receptor-adaptor complex.

[0028]FIG. 13 shows data for a correlation between expression ofHer2-Her3 heterodimers and PI3K//Her3 complexes in tumor cells.

[0029]FIGS. 14A-14B show measurements of Her1-Her2 and Her2-Her3receptor dimer populations obtained from normal breast tissue samplesand from breast tumor tissue samples.

[0030]FIGS. 15A-15G show measurements of Her1-Her1 and Her2-Her2homodimers and Her1-Her2 and Her2-Her3 heterodimers in sections of fixedpellets of cancer cell lines.

DEFINITIONS

[0031] “Antibody” means an immunoglobulin that specifically binds to,and is thereby defined as complementary with, a particular spatial andpolar organization of another molecule. The antibody can be monoclonalor polyclonal and can be prepared by techniques that are well known inthe art such as immunization of a host and collection of sera(polyclonal) or by preparing continuous hybrid cell lines and collectingthe secreted protein (monoclonal), or by cloning and expressingnucleotide sequences or mutagenized versions thereof coding at least forthe amino acid sequences required for specific binding of naturalantibodies. Antibodies may include a complete immunoglobulin or fragmentthereof, which immunoglobulins include the various classes and isotypes,such as IgA, IgD, IgE, IgG1, IgG2a, IgG2b and IgG3, IgM, etc. Fragmentsthereof may include Fab, Fv and F(ab′)2, Fab′, and the like. Inaddition, aggregates, polymers, and conjugates of immunoglobulins ortheir fragments can be used where appropriate so long as bindingaffinity for a particular polypeptide is maintained. Guidance in theproduction and selection of antibodies for use in immunoassays,including such assays employing releasable molecular tag (as describedbelow) can be found in readily available texts and manuals, e.g. Harlowand Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, New York, 1988); Howard and Bethell, Basic Methods in AntibodyProduction and Characterization (CRC Press, 2001); Wild, editor, TheImmunoassay Handbook (Stockton Press, New York, 1994), and the like.

[0032] “Antibody binding composition” means a molecule or a complex ofmolecules that comprises one or more antibodies, or fragments thereof,and derives its binding specificity from such antibody or antibodyfragment. Antibody binding compositions include, but are not limited to,(i) antibody pairs in which a first antibody binds specifically to atarget molecule and a second antibody binds specifically to a constantregion of the first antibody; a biotinylated antibody that bindsspecifically to a target molecule and a streptavidin protein, whichprotein is derivatized with moieties such as molecular tags orphotosensitizers, or the like, via a biotin moiety; (ii) antibodiesspecific for a target molecule and conjugated to a polymer, such asdextran, which, in turn, is derivatized with moieties such as moleculartags or photosensitizers, either directly by covalent bonds orindirectly via streptavidin-biotin linkages; (iii) antibodies specificfor a target molecule and conjugated to a bead, or microbead, or othersolid phase support, which, in turn, is derivatized either directly orindirectly with moieties such as molecular tags or photosensitizers, orpolymers containing the latter.

[0033] “Antigenic determinant,” or “epitope” means a site on the surfaceof a molecule, usually a protein, to which a single antibody moleculebinds; generally a protein has several or many different antigenicdeterminants and reacts with antibodies of many different specificities.A preferred antigenic determinant is a phosphorylation site of aprotein.

[0034] “Binding moiety” means any molecule to which molecular tags canbe directly or indirectly attached that is capable of specificallybinding to an analyte. Binding moieties include, but are not limited to,antibodies, antibody binding compositions, peptides, proteins, nucleicacids, and organic molecules having a molecular weight of up to 1000daltons and consisting of atoms selected from the group consisting ofhydrogen, carbon, oxygen, nitrogen, sulfur, and phosphorus. Preferably,binding moieties are antibodies or antibody binding compositions.

[0035] “Cancer” and “cancerous” refer to or describe the physiologicalcondition in mammals that is typically characterized by unregulated cellgrowth. Examples of cancer include, but are not limited to, carcinoma,lymphoma, blastoma, sarcoma, and leukemia. More particular examples ofsuch cancers include squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, colorectal cancer,endometrial carcinoma, salivary gland carcinoma, kidney cancer, prostatecancer, vulval cancer, thyroid cancer, hepatic carcinoma and varioustypes of head and neck cancer.

[0036] “Complex” as used herein means an assemblage or aggregate ofmolecules in direct or indirect contact with one another. In one aspect,“contact,” or more particularly, “direct contact”in reference to acomplex of molecules, or in reference to specificity or specificbinding, means two or more molecules are close enough so that attractivenoncovalent interactions, such as Van der Waal forces, hydrogen bonding,ionic and hydrophobic interactions, and the like, dominate theinteraction of the molecules. In such an aspect, a complex of moleculesis stable in that under assay conditions the complex isthermodynamically more favorable than a non-aggregated, ornon-complexed, state of its component molecules. As used herein,“complex” usually refers to a stable aggregate of two or more proteins,and is equivalently referred to as a “protein-protein complex.” Mosttypically, a “complex” refers to a stable aggregate of two proteins.

[0037] “Dimer” in reference to cell surface membrane receptors means acomplex of two or more membrane-bound receptor proteins that may be thesame or different. Dimers of identical receptors are referred to as“homodimers” and dimers of different receptors are referred to as“heterodimers.” Dimers usually consist of two receptors in contact withone another. Dimers may be created in a cell surface membrane by passiveprocesses, such as Van der Waal interactions, and the like, as describedabove in the definition of “complex,” or dimers may be created by activeprocesses, such as by ligand-induced dimerization, covalent linkages,interaction with intracellular components, or the like, e.g.Schlessinger, Cell, 103: 211-225 (2000). As used herein, the term“dimer” is understood to refer to “cell surface membrane receptordimer,” unless understood otherwise from the context.

[0038] “Disease status” inlcudes, but is not limited to, the followingfeatures: likelihood of contracting a disease, presence or absence of adisease, prognosis of disease severity, and likelihood that a patientwill respond to treatment by a particular therapeutic agent that actsthrough a receptor complex. In regard to cancer, “disease status”further includes detection of precancerous or cancerous cells ortissues, the selection of patients that are likely to respond totreatment by a therapeutic agent that acts through one or more receptorcomplexes, such as one or more receptor dimers, and the ameliorativeeffects of treatment with such therapeutic agents. In one aspect,disease status in reference to Her receptor complexes means likelihoodthat a cancer patient will respond to treatment by a Her, or ErbB,dimer-acting drug. Preferably, such cancer patient is a breast orovarian cancer patient and such Her dimer-acting drugs include Omnitarg™(2C4), Herceptin, ZD-1839 (Iressa), and OSI-774 (Tarceva).

[0039] “ErbB receptor” or “Her receptor” is a receptor protein tyrosinekinase which belongs to the ErbB receptor family and includes EGFR(“Her1”), ErbB2 (“Her2”), ErbB3 (“Her3”) and ErbB4 (“Her4”) receptors.The ErbB receptor generally comprises an extracellular domain, which maybind an ErbB ligand; a lipophilic transmembrane domain; a conservedintracellular tyrosine kinase domain; and a carboxyl-terminal signalingdomain harboring several tyrosine residues which can be phosphorylated.The ErbB receptor may be a native sequence ErbB receptor or an aminoacid sequence variant thereof. Preferably the ErbB receptor is nativesequence human ErbB receptor. In one aspect, ErbB receptor includestruncated versions of Her receptors, including but not limited to,EGFRvIII and p95Her2, disclosed in Chu et al, Biochem. J., 324: 855-861(1997); Xia et al, Oncogene, 23: 646-653 (2004); and the like.

[0040] The terms “ErbB1”, “epidermal growth factor receptor” and “EGFR”and “Her1” are used interchangeably herein and refer to native sequenceEGFR as disclosed, for example, in Carpenter et al. Ann. Rev. Biochem.56:881-914 (1987), including variants thereof (e.g. a deletion mutantEGFR as in Humphrey et al. PNAS (USA) 87:4207-4211 (1990)). erbB1 refersto the gene encoding the EGFR protein product. Examples of antibodieswhich bind to EGFR include MAb 579 (ATCC CRL RB 8506), MAb 455 (ATCC CRLHB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S.Pat. No. 4,943,533, Mendelsohn et al.) and variants thereof, such aschimerized 225 (C225) and reshaped human 225 (H225) (see, WO 96/40210,Imclone Systems Inc.).

[0041] “Her2”, “ErbB2” “c-Erb-B2” are used interchangeably. Unlessindicated otherwise, the terms “ErbB2” “c-Erb-B2” and “Her2” when usedherein refer to the human protein. The human ErbB2 gene and ErbB2protein are, for example, described in Semba et al., PNAS (USA)82:6497-650 (1985)and Yamamoto et al. Nature 319:230-234 (1986)(Genebank accession number X03363). Examples of antibodies thatspecifically bind to Her2 are disclosed in U.S. Pat. Nos. 5,677,171;5,772,997; Fendly et al, Cancer Res., 50: 1550-1558 (1990); and thelike.

[0042] “ErbB3” and “Her3” refer to the receptor polypeptide asdisclosed, for example, in U.S. Pat. Nos. 5,183,884 and 5,480,968 aswell as Kraus et al. PNAS (USA) 86:9193-9197 (1989), including variantsthereof. Examples of antibodies which bind Her3 are described in U.S.Pat. No. 5,968,511, e.g. the 8B8 antibody (ATCC HB 12070).

[0043] The terms “ErbB4” and “Her4” herein refer to the receptorpolypeptide as disclosed, for example, in EP Pat Appln No 599,274;Plowman et al., Proc. Natl. Acad. Sci. USA, 90:1746-1750 (1993); andPlowman et al., Nature, 366:473-475 (1993), including variants thereofsuch as the Her4 isoforms disclosed in WO 99/19488.

[0044] “Insulin-like growth factor-1 receptor” or “IGF-1R” means a humanreceptor tyrosine kinase substantially identical to those disclosed inUllrich et al, EMBO J., 5: 2503-2512 (1986) or Steele-Perkins et al, J.Biol. Chem., 263: 11486-11492 (1988).

[0045] “Isolated” in reference to a polypeptide or protein meanssubstantially separated from the components of its natural environment.Preferably, an isolated polypeptide or protein is a composition thatconsists of at least eighty percent of the polypeptide or proteinidentified by sequence on a weight basis as compared to components ofits natural environment; more preferably, such composition consists ofat least ninety-five percent of the polypeptide or protein identified bysequence on a weight basis as compared to components of its naturalenvironment; and still more preferably, such composition consists of atleast ninety-nine percent of the polypeptide or protein identified bysequence on a weight basis as compared to components of its naturalenvironment. Most preferably, an isolated polypeptide or protein is ahomogeneous composition that can be resolved as a single spot afterconventional separation by two-dimensional gel electrophoresis based onmolecular weight and isoelectric point. Protocols for such analysis byconventional two-dimensional gel electrophoresis are well known to oneof ordinary skill in the art, e.g. Hames and Rickwood, Editors, GelElectrophoresis of Proteins: A Practical Approach (IRL Press, Oxford,1981); Scopes, Protein Purification (Springer-Verlag, New York, 1982);Rabilloud, Editor, Proteome Research: Two-Dimensional GelElectrophoresis and Identification Methods (Springer-Verlag, Berlin,2000).

[0046] “Kit” refers to any delivery system for delivering materials orreagents for carrying out a method of the invention. In the context ofreaction assays, such delivery systems include systems that allow forthe storage, transport, or delivery of reaction reagents (e.g., probes,enzymes, etc. in the appropriate containers) and/or supporting materials(e.g., buffers, written instructions for performing the assay etc.) fromone location to another. For example, kits include one or moreenclosures (e.g., boxes) containing the relevant reaction reagentsand/or supporting materials. Such contents may be delivered to theintended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains probes.

[0047] “Percent identical,” or like term, used in respect of thecomparison of a reference sequence and another sequence (i.e. a“candidate” sequence) means that in an optimal alignment between the twosequences, the candidate sequence is identical to the reference sequencein a number of subunit positions equivalent to the indicated percentage,the subunits being nucleotides for polynucleotide comparisons or aminoacids for polypeptide comparisons. As used herein, an “optimalalignment” of sequences being compared is one that maximizes matchesbetween subunits and minimizes the number of gaps employed inconstructing an alignment. Percent identities may be determined withcommercially available implementations of algorithms described byNeedleman and Wunsch, J. Mol. Biol., 48: 443-453 (1970)(“GAP” program ofWisconsin Sequence Analysis Package, Genetics Computer Group, Madison,Wis.). Other software packages in the art for constructing alignmentsand calculating percentage identity or other measures of similarityinclude the “BestFit” program, based on the algorithm of Smith andWaterman, Advances in Applied Mathematics, 2: 482-489 (1981) (WisconsinSequence Analysis Package, Genetics Computer Group, Madison, Wis.). Inother words, for example, to obtain a polypeptide having an amino acidsequence at least 95 percent identical to a reference amino acidsequence, up to five percent of the amino acid residues in the referencesequence many be deleted or substituted with another amino acid, or anumber of amino acids up to five percent of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence many occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence of in one or morecontiguous groups with in the references sequence. It is understood thatin making comparisons with reference sequences of the invention thatcandidate sequence may be a component or segment of a larger polypeptideor polynucleotide and that such comparisons for the purpose computingpercentage identity is to be carried out with respect to the relevantcomponent or segment.

[0048] “Phosphatidylinositol 3 kinase protein,” or equivalently a “PI3Kprotein,” means a human intracellular protein of the set of humanproteins describe under NCBI accession numbers NP_(—)852664,NP_(—)852556, and NP_(—)852665, and proteins having amino acid sequencessubstantially identical thereto.

[0049] “Platelet-derived growth factor receptor” or “PDGFR” means ahuman receptor tyrosine kinase protein that is substantially identicalto PDGFRα or PDGFRβ, or variants thereof, described in Heldin et al,Physiological Reviews, 79: 1283-1316 (1999). In one aspect, theinvention includes determining the status of cancers, pre-cancerousconditions, fibrotic or sclerotic conditions by measuring one or moredimers of the following group: PDGFRα homodimers, PDGFRβ homodimers, andPDGFRα-PDGFRβ heterodimers. In particular, fibrotic conditions includelung or kidney fibrosis, and sclerotic conditions includeatherosclerosis. Cancers include, but are not limited to, breast cancer,colorectal carcinoma, glioblastoma, and ovarian carcinoma. Reference to“PDGFR” alone is understood to mean “PDGFRα” or “PDGFRβ.”

[0050] “Polypeptide” refers to a class of compounds composed of aminoacid residues chemically bonded together by amide linkages withelimination of water between the carboxy group of one amino acid and theamino group of another amino acid. A polypeptide is a polymer of aminoacid residues, which may contain a large number of such residues.Peptides are similar to polypeptides, except that, generally, they arecomprised of a lesser number of amino acids. Peptides are sometimesreferred to as oligopeptides. There is no clear-cut distinction betweenpolypeptides and peptides. For convenience, in this disclosure andclaims, the term “polypeptide” will be used to refer generally topeptides and polypeptides. The amino acid residues may be natural orsynthetic.

[0051] “Protein” refers to a polypeptide, usually synthesized by abiological cell, folded into a defined three-dimensional structure.Proteins are generally from about 5,000 to about 5,000,000 or more inmolecular weight, more usually from about 5,000 to about 1,000,000molecular weight, and may include posttranslational modifications, suchacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, farnesylation,demethylation, formation of covalent cross-links, formation of cystine,formation of pyroglutamate, formylation, gamma-carboxylation,glycosylation, GPI anchor formation, hydroxylation, iodination,methylation, myristoylation, oxidation, phosphorylation, prenylation,racemization, selenoylation, sulfation, and ubiquitination, e.g. Wold,F., Post-translational Protein Modifications: Perspectives andProspects, pgs. 1-12 in Post-translational Covalent Modification ofProteins, B. C. Johnson, Ed., Academic Press, New York, 1983. Proteinsinclude, by way of illustration and not limitation, cytokines orinterleukins, enzymes such as, e.g., kinases, proteases, galactosidasesand so forth, protamines, histones, albumins, immunoglobulins,scleroproteins, phosphoproteins, mucoproteins, chromoproteins,lipoproteins, nucleoproteins, glycoproteins, T-cell receptors,proteoglycans, and the like.

[0052] “Reference sample” means one or more cell, xenograft, or tissuesamples that are representative of a normal or non-diseased state towhich measurements on patient samples are compared to determine whethera receptor complex is present in excess or is present in reduced amountin the patient sample. The nature of the reference sample is a matter ofdesign choice for a particular assay and may be derived or determinedfrom normal tissue of the patient him- or herself, or from tissues froma population of healthy individuals. Preferably, values relating toamounts of receptor complexes in reference samples are obtained underessentially identical experimental conditions as corresponding valuesfor patient samples being tested. Reference samples may be from the samekind of tissue as that the patient sample, or it may be from differenttissue types, and the population from which reference samples areobtained may be selected for characteristics that match those of thepatient, such as age, sex, race, and the like. Typically, in assays ofthe invention, amounts of receptor complexes on patient samples arecompared to corresponding values of reference samples that have beenpreviously tabulated and are provided as average ranges, average valueswith standard deviations, or like representations.

[0053] “Receptor complex” means a complex that comprises at least onecell surface membrane receptor. Receptor complexes may include a dimerof cell surface membrane receptors, or one or more intracellularproteins, such as adaptor proteins, that form links in the varioussignaling pathways. Exemplary intracellular proteins that may be part ofa receptor complex includes, but is not limit to, PI3K proteins, Grb2proteins, Grb7 proteins, Shc proteins, and Sos proteins, Src proteins,Cbl proteins, PLCγ proteins, Shp2 proteins, GAP proteins, Nck proteins,Vav proteins, and Crk proteins. In one aspect, receptor complexesinclude PI3K or Shc proteins.

[0054] “Receptor tyrosine kinase,” or “RTK,” means a human receptorprotein having intracellular kinase activity and being selected from theRTK family of proteins described in Schlessinger, Cell, 103: 211-225(2000); and Blume-Jensen and Hunter (cited above). “Receptor tyrosinekinase dimer” means a complex in a cell surface membrane comprising tworeceptor tyrosine kinase proteins. In some aspects, a receptor tyrosinekinase dimer may comprise two covalently linked receptor tyrosine kinaseproteins. Exemplary RTK dimers are listed in Table I. RTK dimers ofparticular interest are Her receptor dimers and VEGFR dimers.

[0055] “Sample” or “tissue sample” or “patient sample” or “patient cellor tissue sample” or “specimen” each means a collection of similar cellsobtained from a tissue of a subject or patient. The source of the tissuesample may be solid tissue as from a fresh, frozen and/or preservedorgan or tissue sample or biopsy or aspirate; blood or any bloodconstituents; bodily fluids such as cerebral spinal fluid, amnioticfluid, peritoneal fluid, or interstitial fluid; or cells from any timein gestation or development of the subject. The tissue sample maycontain compounds which are not naturally intermixed with the tissue innature such as preservatives, anticoagulants, buffers, fixatives,nutrients, antibiotics, or the like. In one aspect of the invention,tissue samples or patient samples are fixed, particularly conventionalformalin-fixed paraffin-embedded samples. Such samples are typicallyused in an assay for receptor complexes in the form of thin sections,e.g. 3-10 μm thick, of fixed tissue mounted on a microscope slide, orequivalent surface. Such samples also typically undergo a conventionalre-hydration procedure, and optionally, an antigen retrieval procedureas a part of, or preliminary to, assay measurements.

[0056] “Separation profile” in reference to the separation of moleculartags means a chart, graph, curve, bar graph, or other representation ofsignal intensity data versus a parameter related to the molecular tags,such as retention time, mass, or the like, that provides a readout, ormeasure, of the number of molecular tags of each type produced in anassay. A separation profile may be an electropherogram, a chromatogram,an electrochromatogram, a mass spectrogram, or like graphicalrepresentation of data depending on the separation technique employed. A“peak” or a “band” or a “zone” in reference to a separation profilemeans a region where a separated compound is concentrated. There may bemultiple separation profiles for a single assay if, for example,different molecular tags have different fluorescent labels havingdistinct emission spectra and data is collected and recorded at multiplewavelengths. In one aspect, released molecular tags are separated bydifferences in electrophoretic mobility to form an electropherogramwherein different molecular tags correspond to distinct peaks on theelectropherogram. A measure of the distinctness, or lack of overlap, ofadjacent peaks in an electropherogram is “electrophoretic resolution,”which may be taken as the distance between adjacent peak maximumsdivided by four times the larger of the two standard deviations of thepeaks. Preferably, adjacent peaks have a resolution of at least 1.0, andmore preferably, at least 1.5, and most preferably, at least 2.0. In agiven separation and detection system, the desired resolution may beobtained by selecting a plurality of molecular tags whose members haveelectrophoretic mobilities that differ by at least a peak-resolvingamount, such quantity depending on several factors well known to thoseof ordinary skill, including signal detection system, nature of thefluorescent moieties, the diffusion coefficients of the tags, thepresence or absence of sieving matrices, nature of the electrophoreticapparatus, e.g. presence or absence of channels, length of separationchannels, and the like. Electropherograms may be analyzed to associatefeatures in the data with the presence, absence, or quantities ofmolecular tags using analysis programs, such as disclosed in Williams etal, U.S. patent publication 2003/0170734 A1.

[0057] “SHC” (standing for “Src homology 2/α-collagen-related”) meansany one of a family of adaptor proteins (66, 52, and 46 kDalton) in RTKsignaling pathways substantially identical to those described in Pelicciet al, Cell, 70: 93-104 (1992). In one aspect, SHC means the humanversions of such adaptor proteins.

[0058] “Signaling pathway” or “signal transduction pathway” means aseries of molecular events usually beginning with the interaction ofcell surface receptor with an extracellular ligand or with the bindingof an intracellular molecule to a phosphorylated site of a cell surfacereceptor that triggers a series of molecular interactions, wherein theseries of molecular interactions results in a regulation of geneexpression in the nucleus of a cell. “Ras-MAPK pathway” means asignaling pathway that includes the phosphorylation of a MAPK proteinsubsequent to the formation of a Ras-GTP complex. “PI3K-Akt pathway”means a signaling pathway that includes the phosphorylation of an Aktprotein by a PI3K protein.

[0059] “Specific” or “specificity” in reference to the binding of onemolecule to another molecule, such as a binding compound, or probe, fora target analyte or complex, means the recognition, contact, andformation of a stable complex between the probe and target, togetherwith substantially less recognition, contact, or complex formation ofthe probe with other molecules. In one aspect, “specific” in referenceto the binding of a first molecule to a second molecule means that tothe extent the first molecule recognizes and forms a complex withanother molecules in a reaction or sample, it forms the largest numberof the complexes with the second molecule. In one aspect, this largestnumber is at least fifty percent of all such complexes form by the firstmolecule. Generally, molecules involved in a specific binding event haveareas on their surfaces or in cavities giving rise to specificrecognition between the molecules binding to each other. Examples ofspecific binding include antibody-antigen interactions, enzyme-substrateinteractions, formation of duplexes or triplexes among polynucleotidesand/or oligonucleotides, receptor-ligand interactions, and the like.

[0060] “Spectrally resolvable” in reference to a plurality offluorescent labels means that the fluorescent emission bands of thelabels are sufficiently distinct, i.e. sufficiently non-overlapping,that molecular tags to which the respective labels are attached can bedistinguished on the basis of the fluorescent signal generated by therespective labels by standard photodetection systems, e.g. employing asystem of band pass filters and photomultiplier tubes, or the like, asexemplified by the systems described in U.S. Pat. Nos. 4,230,558;4,811,218, or the like, or in Wheeless et al, pgs. 21-76, in FlowCytometry: Instrumentation and Data Analysis (Academic Press, New York,1985).

[0061] “Substantially identical” in reference to proteins or amino acidsequences of proteins in a family of related proteins that are beingcompared means either that one protein has an amino acid sequence thatis at least fifty percent identical to the other protein or that oneprotein is an isoform or splice variant of the same gene as the otherprotein. In one aspect, substantially identical means one protein, oramino acid sequence thereof, is at least eighty percent identical to theother protein, or amino acid sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0062] The invention provides a method of using ErbB cell surfacereceptor complexes as biomarkers for the status of a disease or otherphysiological conditions in a biological organism, particularly a cancerstatus in a human. In one aspect, ErbB receptor complexes are measureddirectly from patient samples; that is, measurements are made withoutculturing, formation of xenografts, or the use of like techniques, thatrequire extra labor and expense and that may introduce artifacts and/ornoise into the measurement process. In a particular aspect of theinvention, measurements of one or more receptor complexes are madedirectly on tissue lysates of frozen patient samples or on sections offixed patient samples. In a preferred embodiment, one or more ErbBreceptor complexes are measured in sections of formalin-fixedparaffin-embedded (FFPE) samples.

[0063] In another aspect, the invention provides an indirect measurementof ErbB receptor phosphorylation through the measurement of complexesthat depend on such posttranslational modifications for their formation.

[0064] In one aspect, a plurality of ErbB receptor complexes, such asreceptor dimers, are simultaneously measured in the same assay reactionmixture. Preferably, such complexes are measured using binding compoundshaving one or more molecular tags releasably attached, such that afterbinding to a protein in a complex, the molecular tags may be releasedand separated from the reaction, or assay, mixture for detection and/orquantification.

[0065] In one aspect, the invention provides a method for determining adisease status of a patient comprising the following steps: measuring anamount of each of one or more ErbB receptor dimers in a patient sample;comparing each such amount to its corresponding amount from a referencesample; and correlating differences in the amounts from the patientsample and the respective corresponding amounts from the referencesample to the presence or severity of a disease condition in thepatient. In a preferred embodiment, the step of measuring comprising thesteps of: (i) providing one or more binding compounds specific for aprotein of each of the one or more receptor dimers, such that eachbinding compound has one or more molecular tags each attached thereto bya cleavable linkage, and such that the one or more molecular tagsattached to different binding compounds have different separationcharacteristics so that upon separation molecular tags from differentbinding compounds form distinct peaks in a separation profile; (ii)mixing the binding compounds and the one or more complexes such thatbinding compounds specifically bind to their respective receptor dimersto form detectable complexes; (iii) cleaving the cleavable linkage ofeach binding compound forming detectable complexes, and (iv) separatingand identifying the released molecular tags to determine the presence orabsence or the amount of the one or more receptor dimers.

[0066] In another aspect, the step of measuring the amounts of one ormore types of ErbB receptor dimer comprising the following steps: (i)providing for each of the one or more types of receptor dimer a cleavingprobe specific for a first receptor in each of the one or more receptordimers, each cleaving probe having a cleavage-inducing moiety with aneffective proximity; (ii) providing one or more binding compoundsspecific for a second receptor of each of the one or more receptordimers, such that each binding compound has one or more molecular tagseach attached thereto by a cleavable linkage, and such that the one ormore molecular tags attached to different binding compounds havedifferent separation characteristics so that upon separation moleculartags from different binding compounds form distinct peaks in aseparation profile; (iii) mixing the cleaving probes, the bindingcompounds, and the one or more types of receptor dimers such thatcleaving probes specifically bind to first receptors of the receptordimers and binding compounds specifically bind to the second receptorsof the receptor dimers and such that cleavable linkages of the bindingcompounds are within the effective proximity of cleavage-inducingmoieties of the cleaving probes so that molecular tags are released; and(iv) separating and identifying the released molecular tags to determinethe presence or absence or the amount of the one or more types ofreceptor dimers. Preferably, receptor dimers and first and secondreceptors are selected from the receptor dimers listed in Table I.

[0067] In another aspect of the invention, a biological specimen, whichcomprises a mixed cell population suspected of containing the rare cellof interest is obtained from a patient. A sample is then prepared bymixing the biological specimen with magnetic particles which are coupledto a biospecific ligand specifically reactive with an antigen on therare cell that is different from or not found on blood cells (referredto herein as a “capture antigen”), so that other sample components maybe substantially removed. The sample is subjected to a magnetic fieldwhich is effective to separate cells labeled with the magneticparticles, including the rare cells of interest, if any are present inthe specimen. The cell population so isolated is then analyzed usingmolecular tags conjugated to binding moieties specific for biomarkers todetermine the presence and/or number of rare cells. In a preferredembodiment the magnetic particles used in this method are colloidalmagnetic nanoparticles. Preferably, such rare cell populations arecirculating epithelial cells, which may be isolated from patient's bloodusing epithelial-specific capture antigens, e.g. as disclosed in Hayeset al, International J. of Oncology, 21: 1111-1117 (2002); Soria et al,Clinical Cancer Research, 5: 971-975 (1999); Ady et al, British J.Cancer, 90: 443-448 (2004); which are incorporated by reference. Inparticular, monoclonal antibody BerEP4 (Dynal A. S., Oslo, Norway) maybe used to capture human epithelial cells with magnetic particles.

[0068] In another aspect, the invention provides a method fordetermining a cancer status of a patient comprising the following steps:(i) immunomagnetically isolating a patient sample comprising circulatingepithelial cells by contacting a sample of patient blood with one ormore antibody compositions, each antibody composition being specific fora capture antigen and being attached to a magnetic particle; (ii)measuring an amount of each of one or more ErbB receptor complexes inthe patient sample; comparing each such amount to its correspondingamount from a reference sample; and correlating differences in theamounts from the patient sample and the respective corresponding amountsfrom the reference sample to the presence or severity of a cancercondition in the patient. In a preferred embodiment, the step ofmeasuring comprises the steps of: (i) providing one or more bindingcompounds specific for a protein of each of the one or more ErbBreceptor complexes, such that each binding compound has one or moremolecular tags each attached thereto by a cleavable linkage, and suchthat the one or more molecular tags attached to different bindingcompounds have different separation characteristics so that uponseparation molecular tags from different binding compounds form distinctpeaks in a separation profile; (ii) mixing the binding compounds and theone or more ErbB receptor complexes such that binding compoundsspecifically bind to their respective proteins of the one or more ErbBreceptor complexes to form detectable complexes; (iii) cleaving thecleavable linkage of each binding compound forming detectable complexes,and (iv) separating and identifying the released molecular tags todetermine the presence or absence or the amount of the one or more ErbBreceptor complexes.

[0069] In another aspect, the step of measuring the amounts of one ormore ErbB receptor complexes comprising the following steps: (i)providing for each of the one or more ErbB receptor complexes a cleavingprobe specific for a first protein in each of the one or more ErbBreceptor complexes, each cleaving probe having a cleavage-inducingmoiety with an effective proximity; (ii) providing one or more bindingcompounds specific for a second protein of each of the one or more ErbBreceptor complexes, such that each binding compound has one or moremolecular tags each attached thereto by a cleavable linkage, and suchthat the one or more molecular tags attached to different bindingcompounds have different separation characteristics so that uponseparation molecular tags from different binding compounds form distinctpeaks in a separation profile; (iii) mixing the cleaving probes, thebinding compounds, and the one or more complexes such that cleavingprobes specifically bind to first proteins of the ErbB receptorcomplexes and binding compounds specifically bind to the second proteinsof the ErbB receptor complexes and such that cleavable linkages of thebinding compounds are within the effective proximity ofcleavage-inducing moieties of the cleaving probes so that molecular tagsare released; and (iv) separating and identifying the released moleculartags to determine the presence or absence or the amount of the one ormore ErbB receptor complexes.

Exemplary Receptor Dimer Biomarkers and Dimer-Acting Drugs

[0070] Biomarkers of the invention include dimers and oligomers of thefollowing receptors. TABLE I Exemplary Receptor Complexes of CellSurface Membranes Dimer Dimer Her1-Her1 IGF-1R-Her1 heterodimerHer1-Her2 IGF-1R-Her3 heterodimer Her1-Her3 Her1-PDGFR heterodimersHer1-Her4 Her3-PDGFR heterodimers Her2-Her2 Her2-PI3K Her2-Her3 Her1-SHCHer2-Her4 Her3-SHC Her3-Her4 Her2-SHC Her4-Her4 Her3-PI3K Her2-PDGFRheterodimers Her1-PI3K IGF-1R-Her2 heterodimer

[0071] The mechanisms of action of many drugs that are in use or areunder development require the inhibition of one or more functions ofErbB receptor dimers, such as the association of component receptorsinto a dimer structure, or a function, such as an enzymatic activity,e.g. kinase activity, or autophosphorylation, that depends ondimerization. Such drugs are referred to herein as “dimer-acting” drugs,or “ErbB dimer-acting” drugs. The number, type, formation, and/ordissociation of receptor dimers in the cells of a patient being treated,or whose treatment is contemplated, have a bearing on the effectivenessor suitability of using a particular ErbB dimer-acting drug. Thefollowing ErbB receptor dimers are biomarkers related to the indicateddrugs. In one aspect, the invention provides biomarkers for monitoringthe effect on disease status of an ErbB dimer-acting drug, TABLE IIDrugs Associated with Dimers of Cell Surface Membranes Dimer Drug(s)Her1-Her1, Her1- Cetuximab (Erbitux), Trastuzumab (Herceptin), h- Her2,Her1-Her3, R3 (TheraCIM), ABX-EGF, MDX-447, ZD-1839 Her1-Her4, Her1-(Iressa), OSI-774 (Tarceva), PKI 166, GW572016, IGF-1R, CI-1033,EKB-569, EMD 72000 Her2-IGF-1R Her2-Her1, Her2- 4D4 Mab, Trastuzumab(Herceptin), 2C4, Her3, Her2-Her2, GW572016 Her2-Her4

[0072] The following references describe the dimer-acting drugs listedin Table II: Traxler, Expert Opin. Ther. Targets, 7: 215-234 (2002);Baselga, editor, Oncology Biotherapeutics, 2: 1-36 (2002); Nam et al,Current Drug Targets, 4: 159-179 (2003); Seymour, Current Drug Targets,2: 117-133 (2001); and the like. TABLE III PI3K-Associated ReceptorComplexes Dimer Dimer Her1-Her1 IGF-1R-Her1 heterodimer Her1-Her2Her4-Her4 Her1-Her3 Her3-Her4 Her1-Her4 Her2-Her4 Her2-Her2 Her2-Her3IGF-1R-Her2 heterodimer IGF-1R-Her3 heterodimer Her3-PDGFR heterodimersHer1-PDGFR heterodimers Her2-PDGFR heterodimers Her2-XX-PI3K*

Preparation of Samples

[0073] Samples containing molecular complexes may come from a widevariety of sources for use with the present invention to relate receptorcomplexes populations to disease status or health status, including cellcultures, animal or plant tissues, patient biopsies, or the like.Preferably, samples are human patient samples. Samples are prepared forassays of the invention using conventional techniques, which may dependon the source from which a sample is taken.

[0074] A. Solid Tissue Samples. For biopsies and medical specimens,guidance is provided in the following references: Bancroft JD & StevensA, eds. Theory and Practice of Histological Techniques (ChurchillLivingstone, Edinburgh, 1977); Pearse, Histochemistry. Theory andapplied. 4^(th) ed. (Churchill Livingstone, Edinburgh, 1980).

[0075] In the area of cancerous disease status, examples of patienttissue samples that may be used include, but are not limited to, breast,prostate, ovary, colon, lung, endometrium, stomach, salivary gland orpancreas. The tissue sample can be obtained by a variety of proceduresincluding, but not limited to surgical excision, aspiration or biopsy.The tissue may be fresh or frozen. In one embodiment, assays of theinvention are carried out on tissue samples that have been fixed andembedded in paraffin or the like; therefore, in such embodiments a stepof deparaffination is carried out. A tissue sample may be fixed (i.e.preserved) by conventional methodology [See e.g., “Manual ofHistological Staining Method of the Armed Forces Institute ofPathology,” 3^(rd) edition (1960) Lee G. Luna, HT (ASCP) Editor, TheBlakston Division McGraw-Hill Book Company, New York; The Armed ForcesInstitute of Pathology Advanced Laboratory Methods in Histology andPathology (1994) Ulreka V. Mikel, Editor, Armed Forces Institute ofPathology, American Registry of Pathology, Washington, D.C One of skillin the art will appreciate that the choice of a fixative is determinedby the purpose for which the tissue is to be histologically stained orotherwise analyzed. One of skill in the art will also appreciate thatthe length of fixation depends upon the size of the tissue sample andthe fixative used. By way of example, neutral buffered formalin, Bouin'sor paraformaldehyde, may be used to fix a tissue sample.

[0076] Generally, a tissue sample is first fixed and is then dehydratedthrough an ascending series of alcohols, infiltrated and embedded withparaffin or other sectioning media so that the tissue sample may besectioned. Alternatively, one may section the tissue and fix thesections obtained. By way of example, the tissue sample may be embeddedand processed in paraffin by conventional methodology (See e.g., “Manualof Histological Staining Method of the Armed Forces Institute ofPathology”, supra). Examples of paraffin that may be used include, butare not limited to, Paraplast, Broloid, and Tissuemay. Once the tissuesample is embedded, the sample may be sectioned by a microtome or thelike (See e.g., “Manual of Histological Staining Method of the ArmedForces Institute of Pathology”, supra). By way of example for thisprocedure, sections may have a thickness in a range from about threemicrons to about twelve microns, and preferably, a thickness in a rangeof from about 5 microns to about 10 microns. In one aspect, a sectionmay have an area of from about 10 mm² to about 1 cm². Once cut, thesections may be attached to slides by several standard methods. Examplesof slide adhesives include, but are not limited to, silane, gelatin,poly-L-lysine and the like. By way of example, the paraffin embeddedsections may be attached to positively charged slides and/or slidescoated with poly-L-lysine.

[0077] If paraffin has been used as the embedding material, the tissuesections are generally deparaffinized and rehydrated to water. Thetissue sections may be deparaffinized by several conventional standardmethodologies. For example, xylenes and a gradually descending series ofalcohols may be used (See e.g., “Manual of Histological Staining Methodof the Armed Forces Institute of Pathology”, supra). Alternatively,commercially available deparaffinizing non-organic agents such asHemo-De®) (CMS, Houston, Tex.) may be used.

[0078] For mammalian tissue culture cells, fresh tissues, or likesources, samples may be prepared by conventional cell lysis techniques(e.g. 0.14 M NaCl, 1.5 mM MgCl₂, 10 mM Tris-Cl (pH 8.6), 0.5% NonidetP-40, and protease and/or phosphatase inhibitors as required). For freshmammalian tissues, sample preparation may also include a tissuedisaggregation step, e.g. crushing, mincing, grinding, sonication, orthe like.

[0079] B. Magnetic Isolation of Cells. In some applications, such asmeasuring dimers on rare metastatic cells from a patient's blood, anenrichment step may be carried out prior to conducting an assay forsurface receptor dimer populations. Immunomagnetic isolation orenrichment may be carried out using a variety of techniques andmaterials known in the art, as disclosed in the following representativereferences that are incorporated by reference: Terstappen et al, U.S.Pat. No. 6,365,362; Terstappen et al, U.S. Pat. No. 5,646,001; Rohr etal, U.S. Pat. No. 5,998,224; Kausch et al, U.S. Pat. No. 5,665,582;Kresse et al, U.S. Pat. No. 6,048,515; Kausch et al, U.S. Pat. No.5,508,164; Miltenyi et al, U.S. Pat. No. 5,691,208; Molday, U.S. Pat.No. 4,452,773; Kronick, U.S. Pat. No. 4,375,407; Radbruch et al, chapter23, in Methods in Cell Biology, Vol, 42 (Academic Press, New York,1994); Uhlen et al, Advances in Biomagnetic Separation (EatonPublishing, Natick, 1994); Safarik et al, J. Chromatography B, 722:33-53 (1999); Miltenyi et al, Cytometry, 11: 231-238 (1990); Nakamura etal, Biotechnol. Prog., 17: 1145-1155 (2001); Moreno et al, Urology, 58:386-392 (2001); Racila et al, Proc. Natl. Acad. Sci., 95: 4589-4594(1998); Zigeuner et al, J. Urology, 169: 701-705 (2003); Ghossein et al,Seminars in Surgical Oncology, 20: 304-311 (2001).

[0080] The preferred magnetic particles for use in carrying out thisinvention are particles that behave as colloids. Such particles arecharacterized by their sub-micron particle size, which is generally lessthan about 200 nanometers (nm) (0.20 microns), and their stability togravitational separation from solution for extended periods of time. Inaddition to the many other advantages, this size range makes themessentially invisible to analytical techniques commonly applied to cellanalysis. Particles within the range of 90-150 nm and having between70-90% magnetic mass are contemplated for use in the present invention.Suitable magnetic particles are composed of a crystalline core ofsuperparamagnetic material surrounded by molecules which are bonded,e.g., physically absorbed or covalently attached, to the magnetic coreand which confer stabilizing colloidal properties. The coating materialshould preferably be applied in an amount effective to prevent nonspecific interactions between biological macromolecules found in thesample and the magnetic cores. Such biological macromolecules mayinclude sialic acid residues on the surface of non-target cells,lectins, glyproteins and other membrane components. In addition, thematerial should contain as much magnetic mass/nanoparticle as possible.The size of the magnetic crystals comprising the core is sufficientlysmall that they do not contain a complete magnetic domain. The size ofthe nanoparticles is sufficiently small such that their Brownian energyexceeds their magnetic moment. As a consequence, North Pole, South Polealignment and subsequent mutual attraction/repulsion of these colloidalmagnetic particles does not appear to occur even in moderately strongmagnetic fields, contributing to their solution stability. Finally, themagnetic particles should be separable in high magnetic gradientexternal field separators. That characteristic facilitates samplehandling and provides economic advantages over the more complicatedinternal gradient columns loaded with ferromagnetic beads or steel wool.Magnetic particles having the above-described properties can be preparedby modification of base materials described in U.S. Pat. Nos. 4,795,698,5,597,531 and 5,698,271, which patents are incorporated by reference.

Assays Using Releasable Molecular Tags

[0081] Many advantages are provided by measuring dimer populations usingreleasable molecular tags, including (1) separation of releasedmolecular tags from an assay mixture provides greatly reduced backgroundand a significant gain in sensitivity; and (2) the use of molecular tagsthat are specially designed for ease of separation and detectionprovides a convenient multiplexing capability so that multiple receptorcomplex components may be readily measured simultaneously in the sameassay. Assays employing such tags can have a variety of forms and aredisclosed in the following references: Singh et al, U.S. Pat. No.6,627,400; U.S. patent publications Singh et al, 2002/0013126; and2003/0170915, and Williams et al, 2002/0146726; and Chan-Hui et al,International patent publication WO 2004/011900, all of which areincorporated herein by reference. For example, a wide variety ofseparation techniques may be employed that can distinguish moleculesbased on one or more physical, chemical, or optical differences amongmolecules being separated including but not limited to electrophoreticmobility, molecular weight, shape, solubility, pKa, hydrophobicity,charge, charge/mass ratio, polarity, or the like. In one aspect,molecular tags in a plurality or set differ in electrophoretic mobilityand optical detection characteristics and are separated byelectrophoresis. In another aspect, molecular tags in a plurality or setmay differ in molecular weight, shape, solubility, pKa, hydrophobicity,charge, polarity, and are separated by normal phase or reverse phaseHPLC, ion exchange HPLC, capillary electrochromatography, massspectroscopy, gas phase chromatography, or like technique.

[0082] Sets of molecular tags are provided that are separated intodistinct bands or peaks by a separation technique after they arereleased from binding compounds. Identification and quantification ofsuch peaks provides a measure or profile of the kinds and amounts ofreceptor dimers. Molecular tags within a set may be chemically diverse;however, for convenience, sets of molecular tags are usually chemicallyrelated. For example, they may all be peptides, or they may consist ofdifferent combinations of the same basic building blocks or monomers, orthey may be synthesized using the same basic scaffold with differentsubstituent groups for imparting different separation characteristics,as described more fully below. The number of molecular tags in aplurality may vary depending on several factors including the mode ofseparation employed, the labels used on the molecular tags fordetection, the sensitivity of the binding moieties, the efficiency withwhich the cleavable linkages are cleaved, and the like. In one aspect,the number of molecular tags in a plurality for measuring populations ofreceptor dimers is in the range of from 2 to 10. In other aspects, thesize of the plurality may be in the range of from 2 to 8, 2 to 6, 2 to4, or 2 to 3.

[0083] Receptor dimers may be detected in assays having homogeneousformats or a non-homogeneous, i.e. heterogeneous, formats. In ahomogeneous format, no step is required to separate binding compoundsspecifically bound to target complexes from unbound binding compounds.In a preferred embodiment, homogeneous formats employ reagent pairscomprising (i) one or more binding compounds with releasable moleculartags and (ii) at least one cleaving probe that is capable of generatingan active species that reacts with and releases molecular tags within aneffective proximity of the cleaving probe.

[0084] Receptor dimers may also be detected by assays employing aheterogeneous format. Heterogeneous techniques normally involve aseparation step, where intracellular complexes having binding compoundsspecifically bound are separated from unbound binding compounds, andoptionally, other sample components, such as proteins, membranefragments, and the like. Separation can be achieved in a variety ofways, such as employing a reagent bound to a solid support thatdistinguishes between complex-bound and unbound binding compounds. Thesolid support may be a vessel wall, e.g., microtiter well plate well,capillary, plate, slide, beads, including magnetic beads, liposomes, orthe like. The primary characteristics of the solid support are that it(1) permits segregation of the bound and unbound binding compounds and(2) does not interfere with the formation of the binding complex, or theother operations in the determination of receptor dimers. Usually, infixed samples, unbound binding compounds are removed simply by washing.

[0085] With detection using molecular tags in a heterogeneous format,after washing, a sample may be combined with a solvent into which themolecular tags are to be released. Depending on the nature of thecleavable bond and the method of cleavage, the solvent may include anyadditional reagents for the cleavage. Where reagents for cleavage arenot required, the solvent conveniently may be a separation buffer, e.g.an electrophoretic separation medium. For example, where the cleavablelinkage is photolabile or cleavable via an active species generated by aphotosensitizer, the medium may be irradiated with light of appropriatewavelength to release the molecular tags into the buffer.

[0086] In either format, if the assay reaction conditions interfere withthe separation technique employed, it may be necessary to remove, orexchange, the assay reaction buffer prior to cleavage and separation ofthe molecular tags. For example, in some embodiments, assay conditionsinclude salt concentrations (e.g. required for specific binding) thatdegrade separation performance when molecular tags are separated on thebasis of electrophoretic mobility. In such embodiments, an assay bufferis replaced by a separation buffer, or medium, prior to release andseparation of the molecular tags.

[0087] Assays employing releasable molecular tags and cleaving probescan be made in many different formats and configuations depending on thecomplexes that are detected or measured. Based on the presentdisclosure, it is a design choice for one of ordinary skill in the artto select the numbers and specificities of particular binding compoundsand cleaving probes.

[0088] In one aspect of the invention, the use of releasable moleculartags to measure dimers of cell surface membranes is showndiagrammatically in FIGS. 1A and 1B. Binding compounds (100) havingmolecular tags “mT₁” and “mT₂” and cleaving probe (102) havingphotosensitizer “PS” are combined with biological cells (104). Bindingcompounds having molecular tag “mT₁” are specific for cell surfacereceptors R₁ (106) and binding compounds having molecular tag “mT₂” arespecific for cell surface receptors R₂ (108). Cell surface receptors R₁and R₂ are present as monomers, e.g. (106) and (108), and as dimers(110) in cell surface membrane (112). After these assay components areincubated in a suitable binding buffer to permit the formation (114) ofstable complexes between binding compounds and their respective receptortargets and between the cleaving probe and its receptor target. Asillustrated, preferably binding compounds and cleaving probes eachcomprise an antibody binding composition, which permits the moleculartags and cleavage-inducing moiety to be specifically targeted tomembrane components. In one aspect, such antibody binding compositionsare monoclonal antibodies. In such embodiments, binding buffers maycomprise buffers used in conventional ELISA techniques, or the like.After binding compounds and cleaving probes for stable complexes (116),the assay mixture is illuminated (118) to induce photosensitizers (120)to generate singlet oxygen. Singlet oxygen rapidly reacts withcomponents of the assay mixture so that its effective proximity (122)for cleaving cleavable linkages of molecular tags is spatially limitedso that only molecular tags that happen to be within the effectiveproximity are released (124). As illustrated, the only molecular tagsreleased are those on binding compounds that form stable complexes withR₁-R₂ dimers and a cleaving probe. Released molecular tags (126) areremoved from the assay mixture and separated (128) in accordance with aseparation characteristic so that a distinct peak (130) is formed in aseparation profile (132). In accordance with the invention, such removaland separation may be the same step. Optionally, prior to illuminationthe binding buffer may be removed and replaced with a buffer moresuitable for separation, i.e. a separation buffer. For example, bindingbuffers typically have salt concentrations that may degrade theperformance of some separation techniques, such as capillaryelectrophoresis, for separating molecular tags into distinct peaks. Inone embodiment, such exchange of buffers may be accomplished by membranefiltration.

[0089] An embodiment that illustrates ratiometric measurement ofheterodimers is illustrated in FIG. 1C, in which an additional bindingcompound is employed to give a measure of the total amount of protein(1104) in a sample. Reagents (1122) of the invention comprise (i)cleaving probes (1108), first binding compound (1106), and secondbinding compound (1107), wherein first binding compound (1106) isspecific for protein (1102) and second binding compound (1107) isspecific for protein (1104) at a different antigenic determinant thanthat cleaving probe (1108) is specific for. After binding of thereagents, cleaving probe (1108) is activated to produce active speciesthat cleave the cleavable linkages of the molecular tags within theeffective proximity of the photosensitizer. In this embodiment,molecular tags are released from monomers of protein (1104) that haveboth reagents (1107) and (1108) attached and from heterodimers that havereagent (1108) attached and either or both of reagents (1106) and (1107)attached. Released molecular tags (1123) are separated, and peaks (1118and 1124) in a separation profile (1126) are correlated to the amountsof the released molecular tags. In this embodiment, relative peakheights, or areas, may reflect (i) the differences in affinity of thefirst and second binding compounds for their respective antigenicdeterminants, and/or (ii) the presence or absense of the antigenicdeterminant that the binding compound is specific for. The latersituation is important whenever a binding compound is used to monitorthe post-translational state of a protein, e.g. phosphorylation state.

[0090] Homodimers may be measured as illustrated in FIG. 1D. As above,an assay may comprise three reagents (1128): cleaving probes (1134),first binding compound (1130), and second binding compound (1132). Firstbinding compound (1130) and cleaving probe (1134) are constructed to bespecific for the same antigenic determinant (1135) on protein (1138)that exists (1140) in a sample as either a homodimer (1136) or a monomer(1138). After reagents (1128) are combined with a sample underconditions that promote the formation of stable complexes between thereagents and their respective targets, multiple complexes (1142 through1150) form in the assay mixture. Because cleaving probe (1134) andbinding compound (1130) are specific for the same antigenic determinant(1135), four different combinations (1144 through 1150) of reagents mayform complexes with homodimers. Of the complexes in the assay mixture,only those (1143) with both a cleaving probe (1134) and at least onebinding compound will contribute released molecular tags (1151) forseparation and detection (1154). In this embodiment, the size of peak(1153) is proportional to the amount of homodimer in the assay mixture,while the size of peak (1152) is proportional to the total amount ofprotein (1138) in the assay mixture, both in monomeric form (1142) or inhomodimeric form (1146 and 1148). FIG. 1E illustrates the analogousmeasurements for cell surface receptors that form heterodimers in cellsurface membrane (1161). One skilled in the art would understand thatdimers may be measured in either lysates of cells or tissues, or infixed samples whose membranes have been permeabilized or removed by thefixing process. In such cases, binding compounds may be specific foreither extracellular or intracellular domains of cell surface membranereceptors.

[0091] As illustrated in FIGS. 1E and 1F, releasable molecular tags mayalso be used for the simultaneous detection or measurement of multipledimers and intracellular complexes in a cellular sample. Cells (160),which may be from a sample from in vitro cultures or from a specimen ofpatient tissue, are lysed (172) to render accessable molecular complexesassociated with the cell membrane, and/or post-translationalmodification sites, such as phosphorylation sites, within thecytoplasmic domains of the membrane molecules. After lysing, theresulting lysate (174) is combined with assay reagents (176) thatinclude multiple cleaving probes (175) and multiple binding compounds(177). Assay conditions are selected (178) that allow reagents (176) tospecifically bind to their respective targets, so that upon activationcleavable linkages within the effective proximity (180) of thecleavage-inducing moieties are cleaved and molecular tags are released(182). As above, after cleavage, the released molecular tags areseparated (184) and identified in a separation profile (186), such as anelectropherogram, and based on the number and quantities of moleculartags measured, a profile is obtained of the selected molecular complexesin the cells of the sample.

[0092]FIGS. 1G and 1H illustrate an embodiment of the invention formeasuring receptor complexes in fixed or frozen tissue samples. Fixedtissue sample (1000), e.g. a formalin-fixed paraffin-embedded sample, issliced to provide a section (1004) using a microtome, or likeinstrument, which after placing on surface (1006), which may be amicroscope slide, it is de-waxed and re-hydrated for application ofassay reagents. Enlargement (1007) shows portion (1008) of section(1004) on portion (1014) of microscope slide (1006). Receptor dimermolecules (1018) are illustrated as embedded in the remnants of membranestructure (1016) of the fixed sample. In accordance with this aspect ofthe invention, cleaving probe and binding compounds are incubated withthe fixed sample so that they bind to their target molecules. Forexample, cleaving probes (1012)(illustrated in the figure as an antibodyhaving a photosensitizer (“PS”) attached) and first binding compound(1010)(illustrated as an antibody having molecular tag “mT₁” attached)specifically bind to receptor (1011) common to all of the dimers shown,second binding compound (1017)(with “mT₂”) specifically binds toreceptor (1015), and third binding compound (1019)(with “mT₃”)specifically binds to receptor (1013). After washing to remove bindingcompounds and cleaving probe that are not specifically bound to theirrespective target molecules, buffer (1024) (referred to as “illuminationbuffer” in the figure) is added. For convenience, buffer (1024) may becontained on section (1004), or a portion thereof, by creating ahydrophobic barrier on slide (1006), e.g. with a wax pen. Afterillumination of photosensitizers and release of molecular tags (1026),buffer (1024) now containing release molecular tags (1025) istransferred to a separation device, such as a capillary electrophoresisinstrument, for separation (1028) and identification of the releasedmolecular tags in, for example, electropherogram (1030).

[0093] Measurements made directly on tissue samples, particularly asillustrated in FIGS. 1G and 1H, may be normalized by includingmeasurements on cellular or tissue targets that are representative ofthe total cell number in the sample and/or the numbers of particularsubtypes of cells in the sample. Such tissue targets are referred toherein as “tissue indicators.” The additional measurement may bepreferred, or even necessary, because of the cellular and tissueheterogeneity in patient samples, particularly tumor samples, which maycomprise substantial fractions of normal cells. For example, in FIG. 1H,values for the total amount of receptor (1011) may be given as a ratioof the following two measurements: area of peak (1032) of molecular tag(“mT₁”) and the area of a peak corresponding to a molecular tagcorrelated with a cellular or tissue component common to all the cellsin the sample, e.g. tubulin, or the like. In some cases, where all thecells in the sample are epithelial cells, cytokeratin may be used.Accordingly, detection methods based on releasable molecular tags mayinclude an additional step of providing a binding compound (with adistinct molecular tag) specific for a normalization protein, such astubulin.

[0094]FIGS. 2A-2E illustrate another embodiment of the invention forprofiling dimerization among a plurality of receptor types. FIG. 2Aoutlines the basic steps of such an assay. Cell membranes (200) that areto be tested for dimers of cell surface receptors are combined with setsof binding compounds (202) and (204) and cleaving probe (206). Membranefractions (200) contain three different types of monomer receptormolecules (“1,” “2,” and “3” ) in its cell membrane which associate toform three different heterodimers: 1-2, 1-3, and 2-3. Three antibodyreagents (202) and (204) are combined with membrane fraction (200), eachof the antibody reagents having binding specificity for one of the threereceptor molecules, where antibody (206) is specific for receptormolecule 1, antibody (204) is specific for receptor molecule 2, andantibody (202) is specific for receptor molecule 3. The antibody for thefirst receptor molecule is covalently coupled to a photosensitizermolecule, labeled PS. The antibodies for the second and third receptormolecules are linked to two different tags, labeled T₂ and T₃,respectively, through a linkage that is cleavable by an active speciesgenerated by the photosensitizer moiety.

[0095] After mixing, the antibodies are allowed to bind (208) tomolecules on the surface of the membranes. The photosensitizer isactivated (210), cleaving the linkage between tags and antibodies thatare within an actionable distance from a sensitizer molecule, therebyreleasing tags into the assay medium. Material from the reaction is thenseparated (212), e.g., by capillary electrophoresis, as illustrated. Asshown at the bottom of FIG. 2A, the tags T₂ and T₃ are released, andseparation by electrophoresis will reveal two bands corresponding tothese tags. Because the tags are designed to have a knownelectrophoretic mobility, each of the bands can be uniquely assigned toone of the tags used in the assay.

[0096] As shown in FIG. 2A, only two of the three different heterodimersthat are present in the cell membrane will bind both aphotosensitizer-containing antibody and a tag-containing antibody, andthus only these two species should give rise to released tags. However,multiple experiments are required to measure the relative amounts of thedifferent dimers. FIG. 2B provides a table listing five different assaycombinations. In FIG. 2C are the illustrative results for each assaycomposition. Assay I represents the results from the complete assay, asdescribed in FIG. 2A. In Assay II, the antibody specific for receptormolecule 1, which is linked to the photosensitizer, is omitted. Thisassay yields no signal, indicating that the T₂ and T₃ signals obtainedin Assay I require the photosensitizer reagent. Similarly, Assay V showsthat the tag signals require the presence of the membranes. Assays IIIand IV show that each tagged reagent does not require the presence ofthe other to be cleaved. These results, when considered together, allowone to draw conclusions regarding the presence and composition ofreceptor heterodimers present in the membrane, as given in FIG. 2C,i.e., that both the 1-2 and the 1-3 heterodimer are present.Furthermore, the relative signal intensities from each tag allow one toestimate the relative abundance of each of the heterodimers.

[0097] A conclusion regarding existence of the 2-3 heterodimer cannot bemade with the combination of reagents used in this assay, however. Nosignal representing this complex will be obtained, whether or not thecomplex is present, because it will not have a photosensitizer reagentbound to it. In order to draw conclusions regarding every possibledimeric combination of the three monomers, either a fourth reagent mustbe used that can be localized to every possible oligomer comprisingmonomers 1, 2, and/or 3, or the three binding agents used in thisexperiment must be coupled in different combinations to tags andsensitizer molecules. The later strategy is illustrated in FIGS. 2D and2E. Three possible combinations of photosensitizer and tag distributionamong the three antibody reagents are listed in the table on the left ofFIG. 2D. The first combination comprises a photosensitizer coupled tothe antibody specific for monomer number 1, and is the same combinationused in the illustration of FIG. 2A-2C, and has the same dimerpopulation as in FIG. 2C. The second combination comprises aphotosensitizer coupled to the antibody specific for monomer number 2,and the population profile yields the same number for heterodimer 1-2,plus a value for heterodimer 2-3. The third combination comprises aphotosensitizer coupled to the antibody specific for monomer number 3,and the population profile yields the same number for heterodimer 1-3and 2-3 as obtained from the first two combinations. These results canbe combined to yield the overall heterodimer population profile given inFIG. 2E.

[0098] A preferred embodiment for measuring relative amounts of receptordimers containing a common component receptor is illustrated in FIG. 2F.In this assay design, two different receptor dimers (“1-2” (240) and“2-3” (250)) each having a common component, “2,” may be measuredratiometrically with respect to the common component. An assay design isshown for measuring receptor heterodimer (240) comprising receptor “1”(222) and receptor “2” (220) and receptor heterodimer (250) comprisingreceptor “2” (220) and receptor “3”(224). A key feature of thisembodiment is that cleaving probe (227) be made specific for the commonreceptor of the pair of heterodimers. Binding compound (228) specificfor receptor “2” provides a signal (234) related to the total amount ofreceptor “2” in the assay, whereas binding compound (226) specific forreceptor “1” and binding compound (230) specific for receptor “3”provide signals (232 and 236, respectively) related only to the amountof receptor “1” and receptor “3” present as heterodimers with receptor“2,” respectively. The design of FIG. 2F may be generalized to more thantwo receptor complexes that contain a common component by simply addingbinding compounds specific for the components of the additionalcomplexes.

[0099] A. Binding Compounds

[0100] As mentioned above, mixtures containing pluralities of differentbinding compounds may be provided, wherein each different bindingcompound has one or more molecular tags attached through cleavablelinkages. The nature of the binding compound, cleavable linkage andmolecular tag may vary widely. A binding compound may comprise anantibody binding composition, an antibody, a peptide, a peptide ornon-peptide ligand for a cell surface receptor, a protein, anoligonucleotide, an oligonucleotide analog, such as a peptide nucleicacid, a lectin, or any other molecular entity that is capable ofspecifically binding to a target protein or molecule or stable complexformation with an analyte of interest, such as a complex of proteins. Inone aspect, a binding compound, which can be represented by the formulabelow, comprises one or more molecular tags attached to a bindingmoiety.

B-(L-E)_(k)

[0101] wherein B is binding moiety; L is a cleavable linkage; and E is amolecular tag. In homogeneous assays, cleavable linkage, L, may be anoxidation-labile linkage, and more preferably, it is a linkage that maybe cleaved by singlet oxygen. The moiety “-(L-E)_(k)” indicates that asingle binding compound may have multiple molecular tags attached viacleavable linkages. In one aspect, k is an integer greater than or equalto one, but in other embodiments, k may be greater than several hundred,e.g. 100 to 500, or k is greater than several hundred to as many asseveral thousand, e.g. 500 to 5000. Usually each of the plurality ofdifferent types of binding compound has a different molecular tag, E.Cleavable linkages, e.g. oxidation-labile linkages, and molecular tags,E, are attached to B by way of conventional chemistries.

[0102] Preferably, B is an antibody binding composition thatspecifically binds to a target, such as a predetermined antigenicdeterminant of a target protein, such as a cell surface receptor. Suchcompositions are readily formed from a wide variety of commerciallyavailable antibodies, either monoclonal and polyclonal, specific forproteins of interest. In particular, antibodies specific for epidermalgrowth factor receptors are disclosed in the following patents, whichare incorporated by references: U.S. Pat. Nos. 5,677,171; 5,772,997;5,968,511; 5,480,968; 5,811,098. U.S. Pat. No. 6,488,390, incorporatedherein by reference, discloses antibodies specific for a G-proteincoupled receptor, CCR4. U.S. Pat. No. 5,599,681, incorporated herein byreference, discloses antibodies specific for phosphorylation sites ofproteins. Commercial vendors, such as Cell Signaling Technology(Beverly, Mass.), Biosource International (Camarillo, Calif.), andUpstate (Charlottesville, Va.), also provide monoclonal and polyclonalantibodies specific for many receptors.

[0103] Cleavable linkage, L, can be virtually any chemical linking groupthat may be cleaved under conditions that do not degrade the structureor affect detection characteristics of the released molecular tag, E.Whenever a cleaving probe is used in a homogeneous assay format,cleavable linkage, L, is cleaved by a cleavage agent generated by thecleaving probe that acts over a short distance so that only cleavablelinkages in the immediate proximity of the cleaving probe are cleaved.Typically, such an agent must be activated by making a physical orchemical change to the reaction mixture so that the agent produces ashort lived active species that diffuses to a cleavable linkage toeffect cleavage. In a homogeneous format, the cleavage agent ispreferably attached to a binding moiety, such as an antibody, thattargets prior to activation the cleavage agent to a particular site inthe proximity of a binding compound with releasable molecular tags. Insuch embodiments, a cleavage agent is referred to herein as a“cleavage-inducing moiety,” which is discussed more fully below.

[0104] In a non-homogeneous format, because specifically bound bindingcompounds are separated from unbound binding compounds, a widerselection of cleavable linkages and cleavage agents are available foruse. Cleavable linkages may not only include linkages that are labile toreaction with a locally acting reactive species, such as hydrogenperoxide, singlet oxygen, or the like, but also linkages that are labileto agents that operate throughout a reaction mixture, such asbase-labile linkages, photocleavable linkages, linkages cleavable byreduction, linkages cleaved by oxidation, acid-labile linkages, peptidelinkages cleavable by specific proteases, and the like. Referencesdescribing many such linkages include Greene and Wuts, Protective Groupsin Organic Synthesis, Second Edition (John Wiley & Sons, New York,1991); Hermanson, Bioconjugate Techniques (Academic Press, New York,1996); and Still et al, U.S. Pat. No. 5,565,324.

[0105] In one aspect, commercially available cleavable reagent systemsmay be employed with the invention. For example, a disulfide linkage maybe introduced between an antibody binding composition and a moleculartag using a heterofunctional agent such as N-succinimidyl3-(2-Pyridyldithio)propionate (SPDP),succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene (SMPT), orthe like, available from vendors such as Pierce Chemical Company(Rockford, Ill.). Disulfide bonds introduced by such linkages can bebroken by treatment with a reducing agent, such as dithiothreitol (DTT),dithioerythritol (DTE), 2-mercaptoethanol, sodium borohydride, or thelike. Typical concentrations of reducing agents to effect cleavage ofdisulfide bonds are in the range of from 10 to 100 mM. An oxidativelylabile linkage may be introduced between an antibody binding compositionand a molecular tag using the homobifunctional NHS ester cross-linkingreagent, disuccinimidyl tartarate (DST)(available from Pierce) thatcontains central cis-diols that are susceptible to cleavage with sodiumperiodate (e.g., 15 mM periodate at physiological pH for 4 hours).Linkages that contain esterified spacer components may be cleaved withstrong nucleophilic agents, such as hydroxylamine, e.g. 0.1 Nhydroxylamine, pH 8.5, for 3-6 hours at 37° C. Such spacers can beintroduced by a homobifunctional cross-linking agent such as ethyleneglycol bis(succinimidylsuccinate)(EGS) available from Pierce (Rockford,Ill.). A base labile linkage can be introduced with a sulfone group.Homobifunctional cross-linking agents that can be used to introducesulfone groups in a cleavable linkage includebis[2-(succinimidyloxycarbonyloxy)ethyl]sulfone (BSOCOES), and4,4-difluoro-3,3-dinitrophenylsulfone (DFDNPS). Exemplary basicconditions for cleavage include 0.1 M sodium phosphate, adjusted to pH11.6 by addition of Tris base, containing 6 M urea, 0.1% SDS, and 2 mMDTT, with incubation at 37° C. for 2 hours. Photocleavable linkagesinclude those disclosed in Rothschild et al, U.S. Pat. No. 5,986,076.

[0106] When L is oxidation labile, L may be a thioether or its seleniumanalog; or an olefin, which contains carbon-carbon double bonds, whereincleavage of a double bond to an oxo group, releases the molecular tag,E. Illustrative oxidation labile linkages are disclosed in Singh et al,U.S. Pat. No. 6,627,400; and U.S. patent publications Singh et al,2002/0013126; and 2003/0170915, and in Willner et al, U.S. Pat. No.5,622,929, all of which are incorporated herein by reference.

[0107] Molecular tag, E, in the present invention may comprise anelectrophoric tag as described in the following references whenseparation of pluralities of molecular tags are carried out by gaschromatography or mass spectrometry: Zhang et al, Bioconjugate Chem.,13: 1002-1012 (2002); Giese, Anal. Chem., 2: 165-168 (1983); and U.S.Pat. Nos. 4,650,750; 5,360,819; 5,516,931; 5,602,273; and the like.

[0108] Molecular tag, E, is preferably a water-soluble organic compoundthat is stable with respect to the active species, especially singletoxygen, and that includes a detection or reporter group. Otherwise, Emay vary widely in size and structure. In one aspect, E has a molecularweight in the range of from about 50 to about 2500 daltons, morepreferably, from about 50 to about 1500 daltons. Preferred structures ofE are described more fully below. E may comprise a detection group forgenerating an electrochemical, fluorescent, or chromogenic signal. Inembodiments employing detection by mass, E may not have a separatemoiety for detection purposes. Preferably, the detection group generatesa fluorescent signal.

[0109] Molecular tags within a plurality are selected so that each has aunique separation characteristic and/or a unique optical property withrespect to the other members of the same plurality. In one aspect, thechromatographic or electrophoretic separation characteristic isretention time under set of standard separation conditions conventionalin the art, e.g. voltage, column pressure, column type, mobile phase,electrophoretic separation medium, or the like. In another aspect, theoptical property is a fluorescence property, such as emission spectrum,fluorescence lifetime, fluorescence intensity at a given wavelength orband of wavelengths, or the like. Preferably, the fluorescence propertyis fluorescence intensity. For example, each molecular tag of aplurality may have the same fluorescent emission properties, but eachwill differ from one another by virtue of a unique retention time. Onthe other hand, or two or more of the molecular tags of a plurality mayhave identical migration, or retention, times, but they will have uniquefluorescent properties, e.g. spectrally resolvable emission spectra, sothat all the members of the plurality are distinguishable by thecombination of molecular separation and fluorescence measurement.

[0110] Preferably, released molecular tags are detected byelectrophoretic separation and the fluorescence of a detection group. Insuch embodiments, molecular tags having substantially identicalfluorescence properties have different electrophoretic mobilities sothat distinct peaks in an electropherogram are formed under separationconditions. Preferably, pluralities of molecular tags of the inventionare separated by conventional capillary electrophoresis apparatus,either in the presence or absence of a conventional sieving matrix.Exemplary capillary electrophoresis apparatus include Applied Biosystems(Foster City, Calif.) models 310, 3100 and 3700; Beckman (Fullerton,Calif.) model P/ACE MDQ; Amersham Biosciences (Sunnyvale, Calif.)MegaBACE 1000 or 4000; SpectruMedix genetic analysis system; and thelike. Electrophoretic mobility is proportional to q/M^(2/3), where q isthe charge on the molecule and M is the mass of the molecule. Desirably,the difference in mobility under the conditions of the determinationbetween the closest electrophoretic labels will be at least about 0.001,usually 0.002, more usually at least about 0.01, and may be 0.02 ormore. Preferably, in such conventional apparatus, the electrophoreticmobilities of molecular tags of a plurality differ by at least onepercent, and more preferably, by at least a percentage in the range offrom 1 to 10 percent. Molecular tags are identified and quantified byanalysis of a separation profile, or more specifically, anelectropherogram, and such values are correlated with the amounts andkinds of receptor dimers present in a sample. For example, during orafter electrophoretic separation, the molecular tags are detected oridentified by recording fluorescence signals and migration times (ormigration distances) of the separated compounds, or by constructing achart of relative fluorescent and order of migration of the moleculartags (e.g., as an electropherogram). Preferably, the presence, absence,and/or amounts of molecular tags are measured by using one or morestandards as disclosed by Williams et al, U.S. patent publication2003/0170734A1, which is incorporated herein by reference.

[0111] Pluralities of molecular tags may also be designed for separationby chromatography based on one or more physical characteristics thatinclude but are not limited to molecular weight, shape, solubility, pKa,hydrophobicity, charge, polarity, or the like, e.g. as disclosed in U.S.patent publication 2003/0235832, which is incorporated by reference. Achromatographic separation technique is selected based on parameterssuch as column type, solid phase, mobile phase, and the like, followedby selection of a plurality of molecular tags that may be separated toform distinct peaks or bands in a single operation. Several factorsdetermine which HPLC technique is selected for use in the invention,including the number of molecular tags to be detected (i.e. the size ofthe plurality), the estimated quantities of each molecular tag that willbe generated in the assays, the availability and ease of synthesizingmolecular tags that are candidates for a set to be used in multiplexedassays, the detection modality employed, and the availability,robustness, cost, and ease of operation of HPLC instrumentation,columns, and solvents. Generally, columns and techniques are favoredthat are suitable for analyzing limited amounts of sample and thatprovide the highest resolution separations. Guidance for making suchselections can be found in the literature, e.g. Snyder et al, PracticalHPLC Method Development, (John Wiley & Sons, New York, 1988); Millner,“High Resolution Chromatography: A Practical Approach”, OxfordUniversity Press, New York (1999), Chi-San Wu, “Column Handbook for SizeExclusion Chromatography”, Academic Press, San Diego (1999), and Oliver,“HPLC of Macromolecules: A Practical Approach, Oxford University Press”,Oxford, England (1989). In particular, procedures are available forsystematic development and optimization of chromatographic separationsgiven conditions, such as column type, solid phase, and the like, e.g.Haber et al, J. Chromatogr. Sci., 38: 386-392 (2000); Outinen et al,Eur. J. Pharm. Sci., 6: 197-205 (1998); Lewis et al, J. Chromatogr.,592: 183-195 and 197-208 (1992); and the like. An exemplary HPLCinstrumentation system suitable for use with the present invention isthe Agilent 1100 Series HPLC system (Agilent Technologies, Palo Alto,Calif.).

[0112] In one aspect, molecular tag, E, is (M, D), where M is amobility-modifying moiety and D is a detection moiety. The notation “(M,D)” is used to indicate that the ordering of the M and D moieties may besuch that either moiety can be adjacent to the cleavable linkage, L.That is, “B-L-(M, D)” designates binding compound of either of twoforms: “B-L-M-D” or “B-L-D-M.”

[0113] Detection moiety, D, may be a fluorescent label or dye, achromogenic label or dye, an electrochemical label, or the like.Preferably, D is a fluorescent dye. Exemplary fluorescent dyes for usewith the invention include water-soluble rhodamine dyes, fluoresceins,4,7-dichloroflouresceins, benzoxanthene dyes, and energy transfer dyes,disclosed in the following references: Handbook of Molecular Probes andResearch Reagents, 8^(th) ed., (Molecular Probes, Eugene, 2002); Lee etal, U.S. Pat. No. 6,191,278; Lee et al, U.S. Pat. No. 6,372,907; Menchenet al, U.S. Pat. No. 6,096,723; Lee et al, U.S. Pat. No. 5,945,526; Leeet al, Nucleic Acids Research, 25: 2816-2822 (1997); Hobb, Jr., U.S.Pat. No. 4,997,928; Khanna et al., U.S. Pat. No. 4,318,846; and thelike. Preferably, D is a fluorescein or a fluorescein derivative.

[0114] In an embodiment illustrated in FIG. 3A, binding compoundscomprise a biotinylated antibody (300) as a binding moiety. Moleculartags are attached to binding moiety (300) by way of avidin orstreptavidin bridge (306). Preferably, in operation, binding moiety(300) is first reacted with a target complex, after which avidin orstreptavidin is added (304) to form antibody-biotin-avidin biotin-avidincomplex (305). To such complexes (305) are added (308) biotinylatedmolecular tags (310) to form binding compound (312).

[0115] In still another embodiment illustrated in FIG. 3B, bindingcompounds comprise an antibody (314) derivatized with a multi-functionalmoiety (316) that contains multiple functional groups (318) that arereacted (320) molecular tag precursors to give a final binding compoundhaving multiple molecular tags (322) attached. Exemplarymulti-functional moieties include aminodextran, and like materials.

[0116] Once each of the binding compounds is separately derivatized by adifferent molecular tag, it is pooled with other binding compounds toform a plurality of binding compounds. Usually, each different kind ofbinding compound is present in a composition in the same proportion;however, proportions may be varied as a design choice so that one or asubset of particular binding compounds are present in greater or lowerproportion depending on the desirability or requirements for aparticular embodiment or assay. Factors that may affect such designchoices include, but are not limited to, antibody affinity and avidityfor a particular target, relative prevalence of a target, fluorescentcharacteristics of a detection moiety of a molecular tag, and the like.

[0117] B. Cleavage-Inducing Moiety Producing Active Species

[0118] A cleavage-inducing moiety, or cleaving agent, is a group thatproduces an active species that is capable of cleaving a cleavablelinkage, preferably by oxidation. Preferably, the active species is achemical species that exhibits short-lived activity so that itscleavage-inducing effects are only in the proximity of the site of itsgeneration. Either the active species is inherently short lived, so thatit will not create significant background because beyond the proximityof its creation, or a scavenger is employed that efficiently scavengesthe active species, so that it is not available to react with cleavablelinkages beyond a short distance from the site of its generation.Illustrative active species include singlet oxygen, hydrogen peroxide,NADH, and hydroxyl radicals, phenoxy radical, superoxide, and the like.Illustrative quenchers for active species that cause oxidation includepolyenes, carotenoids, vitamin E, vitamin C, amino acid-pyrroleN-conjugates of tyrosine, histidine, and glutathione, and the like, e.g.Beutner et al, Meth. Enzymol., 319: 226-241 (2000).

[0119] An important consideration in designing assays employing acleavage-inducing moiety and a cleavable linkage is that they not be sofar removed from one another when bound to a receptor complex that theactive species generated by the cleavage-inducing moiety cannotefficiently cleave the cleavable linkage. In one aspect, cleavablelinkages preferably are within 1000 nm, and preferably within 20-200 nm,of a bound cleavage-inducing moiety. More preferably, forphotosensitizer cleavage-inducing moieties generating singlet oxygen,cleavable linkages are within about 20-100 nm of a photosensitizer in areceptor complex. The range within which a cleavage-inducing moiety caneffectively cleave a cleavable linkage (that is, cleave enough moleculartag to generate a detectable signal) is referred to herein as its“effective proximity.” One of ordinary skill in the art recognizes thatthe effective proximity of a particular sensitizer may depend on thedetails of a particular assay design and may be determined or modifiedby routine experimentation.

[0120] A sensitizer is a compound that can be induced to generate areactive intermediate, or species, usually singlet oxygen. Preferably, asensitizer used in accordance with the invention is a photosensitizer.Other sensitizers included within the scope of the invention arecompounds that on excitation by heat, light, ionizing radiation, orchemical activation will release a molecule of singlet oxygen. The bestknown members of this class of compounds include the endoperoxides suchas 1,4-biscarboxyethyl-1,4-naphthalene endoperoxide,9,10-diphenylanthracene-9,10-endoperoxide and 5,6,11,12-tetraphenylnaphthalene 5,12-endoperoxide. Heating or direct absorption of light bythese compounds releases singlet oxygen. Further sensitizers aredisclosed in the following references: Di Mascio et al, FEBS Lett., 355:287 (1994)(peroxidases and oxygenases); Kanofsky, J.Biol. Chem. 258:5991-5993 (1983)(lactoperoxidase); Pierlot et al, Meth. Enzymol., 319:3-20 (2000)(thermal lysis of endoperoxides); and the like. Attachment ofa binding agent to the cleavage-inducing moiety may be direct orindirect, covalent or non-covalent and can be accomplished by well-knowntechniques, commonly available in the literature. See, for example,“Immobilized Enzymes,” Ichiro Chibata, Halsted Press, New York (1978);Cuatrecasas, J. Biol. Chem., 245:3059 (1970).

[0121] As mentioned above, the preferred cleavage-inducing moiety inaccordance with the present invention is a photosensitizer that producessinglet oxygen. As used herein, “photosensitizer” refers to alight-adsorbing molecule that when activated by light converts molecularoxygen into singlet oxygen. Photosensitizers may be attached directly orindirectly, via covalent or non-covalent linkages, to the binding agentof a class-specific reagent. Guidance for constructing of suchcompositions, particularly for antibodies as binding agents, availablein the literature, e.g. in the fields of photodynamic therapy,immunodiagnostics, and the like. The following are exemplary references:Ullman, et al., Proc. Natl. Acad. Sci. USA 91, 5426-5430 (1994); Stronget al, Ann. New York Acad. Sci., 745: 297-320 (1994); Yarmush et al,Crit. Rev. Therapeutic Drug Carrier Syst., 10: 197-252 (1993); Pease etal, U.S. Pat. No. 5,709,994; Ullman et al, U.S. Pat. No. 5,340,716;Ullman et al, U.S. Pat. No. 6,251,581; McCapra, U.S. Pat. No. 5,516,636;and the like.

[0122] A large variety of light sources are available to photo-activatephotosensitizers to generate singlet oxygen. Both polychromatic andmonchromatic sources may be used as long as the source is sufficientlyintense to produce enough singlet oxygen in a practical time duration.The length of the irradiation is dependent on the nature of thephotosensitizer, the nature of the cleavable linkage, the power of thesource of irradiation, and its distance from the sample, and so forth.In general, the period for irradiation may be less than about amicrosecond to as long as about 10 minutes, usually in the range ofabout one millisecond to about 60 seconds. The intensity and length ofirradiation should be sufficient to excite at least about 0.1 % of thephotosensitizer molecules, usually at least about 30% of thephotosensitizer molecules and preferably, substantially all of thephotosensitizer molecules. Exemplary light sources include, by way ofillustration and not limitation, lasers such as, e.g., helium-neonlasers, argon lasers, YAG lasers, He/Cd lasers, and ruby lasers;photodiodes; mercury, sodium and xenon vapor lamps; incandescent lampssuch as, e.g., tungsten and tungsten/halogen; flashlamps; and the like.By way of example, a photoactivation device disclosed in Bjornson et al,International patent publication WO 03/051669 is employed. Briefly, thephotoactivation device is an array of light emitting diodes (LEDs)mounted in housing that permits the simultaneous illumination of all thewells in a 96-well plate. A suitable LED for use in the presentinvention is a high power GaAIAs IR emitter, such as model OD-880Wmanufactured by OPTO DIODE CORP. (Newbury Park, Calif.).

[0123] Examples of photosensitizers that may be utilized in the presentinvention are those that have the above properties and are enumerated inthe following references: Singh and Ullman, U.S. Pat. No. 5,536,834; Liet al, U.S. Pat. No. 5,763,602; Martin et al, Methods Enzymol., 186:635-645 (1990); Yarmush et al, Crit. Rev. Therapeutic Drug CarrierSyst., 10: 197-252 (1993); Pease et al, U.S. Pat. No. 5,709,994; Ullmanet al, U.S. Pat. No. 5,340,716; Ullman et al, U.S. Pat. No. 6,251,581;McCapra, U.S. Pat. No. 5,516,636; Thetford, European patent publ.0484027; Sessler et al, SPIE, 1426: 318-329 (1991); Magda et al, U.S.Pat. No. 5,565,552; Roelant, U.S. Pat. No. 6,001,673; and the like.

[0124] As with sensitizers, in certain embodiments, a photosensitizermay be associated with a solid phase support by being covalently ornon-covalently attached to the surface of the support or incorporatedinto the body of the support. In general, the photosensitizer isassociated with the support in an amount necessary to achieve thenecessary amount of singlet oxygen. Generally, the amount ofphotosensitizer is determined empirically.

[0125] In one embodiment, a photosensitizer is incorporated into a latexparticle to form photosensitizer beads, e.g. as disclosed by Pease etal., U.S. Pat. No. 5,709,994; Pollner, U.S. Pat. No. 6,346,384; andPease et al, PCT publication WO 01/84157. Alternatively, photosensitizerbeads may be prepared by covalently attaching a photosensitizer, such asrose bengal, to 0.5 micron latex beads by means of chloromethyl groupson the latex to provide an ester linking group, as described in J. Amer.Chem. Soc., 97: 3741 (1975). Use of such photosensitizer beads isillustrated in FIG. 3C. As described in FIG. 1C for heteroduplexdetection, complexes (330) are formed after combining reagents (1122)with a sample. This reaction may be carried out, for example, in aconventional 96-well or 384-well microtiter plate, or the like, having afilter membrane that forms one wall, e.g. the bottom, of the wells thatallows reagents to be removed by the application of a vacuum. Thisallows the convenient exchange of buffers, if the buffer required forspecific binding of binding compounds is different that the bufferrequired for either singlet oxygen generation or separation. Forexample, in the case of antibody-based binding compounds, a high saltbuffer is required. If electrophoretic separation of the released tagsis employed, then better performance is achieved by exchanging thebuffer for one that has a lower salt concentration suitable forelectrophoresis. In this embodiment, instead of attaching aphotosensitizer directly to a binding compound, such as an antibody, acleaving probe comprises two components: antibody (332) derivatized witha capture moiety, such as biotin (indicated in FIG. 3C as “bio”) andphotosensitizer bead (338) whose surface is derivatized with an agent(334) that specifically binds with the capture moiety, such as avidin orstreptavidin. Complexes (330) are then captured (335) by photosensitizerbeads by way of the capture moiety, such as biotin (336). Conveniently,if the pore diameter of the filter membrane is selected so thatphotosensitizer beads (338) cannot pass, then a buffer exchange alsoserves to remove unbound binding compounds, which leads to an improvedsignal. After an appropriate buffer for separation has been added, ifnecessary, photosensitizer beads (338) are illuminated so that singletoxygen is generated (342) and molecular tags are released (344). Suchreleased molecular tags (346) are then separated to form separationprofile (352) and dimers are quantified ratiometrically from peaks (348)and (350). Photosensitizer beads may be used in either homogeneous orheterogeneous assay formats.

[0126] Preferably, when analytes, such as cell surface receptors, arebeing detected or antigen in a fixed sample, a cleaving probe maycomprise a primary haptenated antibody and a secondary anti-haptenbinding protein derivatized with multiple photosensitizer molecules. Apreferred primary haptenated antibody is a biotinylated antibody, andpreferred secondary anti-hapten binding proteins may be either ananti-biotin antibody or streptavidin. Other combinations of such primaryand secondary reagents are well known in the art, e.g. Haugland,Handbook of Fluorescent Probes and Research Reagents, Ninth Edition(Molecular Probes, Eugene, Oreg., 2002). An exemplary combination ofsuch reagents is illustrated in FIG. 3E. There binding compounds (366and 368) having releasable tags (“mT₁” and “mT₂” in the Figure), andprimary antibody (368) derivatized with biotin (369) are specificallybound to different epitopes of receptor dimer (362) in membrane (360).Biotin-specific binding protein (370), e.g. streptavidin, is attached tobiotin (369) bringing multiple photosensitizers (372) into effectiveproximity of binding compounds (366 and 368). Biotin-specific bindingprotein (370) may also be an anti-biotin antibody, and photosensitizersmay be attached via free amine group on the protein by conventionalcoupling chemistries, e.g. Hermanson (cited above). An exemplaryphotosensitizer for such use is an NHS ester of methylene blue preparedas disclosed in Shimadzu et al, European patent publication 0510688.

Assay Conditions

[0127] The following general discussion of methods and specificconditions and materials are by way of illustration and not limitation.One of ordinary skill in the art will understand how the methodsdescribed herein can be adapted to other applications, particularly withusing different samples, cell types and target complexes.

[0128] In conducting the methods of the invention, a combination of theassay components is made, including the sample being tested, the bindingcompounds, and optionally the cleaving probe. Generally, assaycomponents may be combined in any order. In certain applications,however, the order of addition may be relevant. For example, one maywish to monitor competitive binding, such as in a quantitative assay. Orone may wish to monitor the stability of an assembled complex. In suchapplications, reactions may be assembled in stages, and may requireincubations before the complete mixture has been assembled, or beforethe cleaving reaction is initiated.

[0129] The amounts of each reagent are usually determined empirically.The amount of sample used in an assay will be determined by thepredicted number of target complexes present and the means of separationand detection used to monitor the signal of the assay. In general, theamounts of the binding compounds and the cleaving probe are provided inmolar excess relative to the expected amount of the target molecules inthe sample, generally at a molar excess of at least 1.5, more desirablyabout 10-fold excess, or more. In specific applications, theconcentration used may be higher or lower, depending on the affinity ofthe binding agents and the expected number of target molecules presenton a single cell. Where one is determining the effect of a chemicalcompound on formation of oligomeric cell surface complexes, the compoundmay be added to the cells prior to, simultaneously with, or afteraddition of the probes, depending on the effect being monitored.

[0130] The assay mixture is combined and incubated under conditions thatprovide for binding of the probes to the cell surface molecules, usuallyin an aqueous medium, generally at a physiological pH (comparable to thepH at which the cells are cultures), maintained by a buffer at aconcentration in the range of about 10 to 200 mM. Conventional buffersmay be used, as well as other conventional additives as necessary, suchas salts, growth medium, stabilizers, etc. Physiological and constanttemperatures are normally employed. Incubation temperatures normallyrange from about 4° to 70° C., usually from about 15° to 45° C., moreusually 25° to 37°.

[0131] After assembly of the assay mixture and incubation to allow theprobes to bind to cell surface molecules, the mixture is treated toactivate the cleaving agent to cleave the tags from the bindingcompounds that are within the effective proximity of the cleaving agent,releasing the corresponding tag from the cell surface into solution. Thenature of this treatment will depend on the mechanism of action of thecleaving agent. For example, where a photosensitizer is employed as thecleaving agent, activation of cleavage will comprise irradiation of themixture at the wavelength of light appropriate to the particularsensitizer used.

[0132] Following cleavage, the sample is then analyzed to determine theidentity of tags that have been released. Where an assay employing aplurality of binding compounds is employed, separation of the releasedtags will generally precede their detection. The methods for bothseparation and detection are determined in the process of designing thetags for the assay. A preferred mode of separation employselectrophoresis, in which the various tags are separated based on knowndifferences in their electrophoretic mobilities.

[0133] As mentioned above, in some embodiments, if the assay reactionconditions may interfere with the separation technique employed, it maybe necessary to remove, or exchange, the assay reaction buffer prior tocleavage and separation of the molecular tags. For example, assayconditions may include salt concentrations (e.g. required for specificbinding) that degrade separation performance when molecular tags areseparated on the basis of electrophoretic mobility. Thus, such high saltbuffers may be removed, e.g. prior to cleavage of molecular tags, andreplaced with another buffer suitable for electrophoretic separationthrough filtration, aspiration, dilution, or other means.

EXAMPLES Sources of Materials Used in Examples

[0134] Antibodies specific for Her receptors, adaptor molecules, andnormalization standards are obtained from commercial vendors, includingLabvision, Cell Signaling Technology, and BD Biosciences. All cell lineswere purchased from ATCC. All human snap-frozen tissue samples werepurchased from either William Bainbridge Genome Foundation (Seattle,Wash.) or Bio Research Support (Boca Raton, Fla.) and were approved byInstitutional Research Board (IRB) at the supplier.

[0135] The molecular tag-antibody conjugates used below are formed byreacting NHS esters of the molecular tag with a free amine on theindicated antibody using conventional procedures. Molecular tags,identified below by their “Pro_N” designations, are disclosed in thefollowing references: Singh et al, U.S. patent publications, 2003/017915and 2002/0013126, which are incorporated by reference. Briefly, bindingcompounds below are molecular tag-monoclonal antibody conjugates formedby reacting an NHS ester of a molecular tag with free amines of theantibodies in a conventional reaction.

Example 1 Analysis of Cell Lysates for Her-2 Heterodimerization andReceptor Phosphorylation

[0136] In this example, Her1-Her2 and Her2-Her3 heterodimers andphosphorylation states are measured in cell lysates from several celllines after treatment with various concentrations of epidermal growthfactor (EGF) and heregulin (HRG). Measurements are made using threebinding compounds and a cleaving probe as described below.

[0137] Sample Preparation:

[0138] 1. Serum-starve breast cancer cell line culture overnight beforeuse.

[0139] 2. Stimulate cell lines with EGF and/or HRG in culture media for10 minutes at 37° C. Exemplary doses of EGF/HRG are 0, 0.032, 0.16, 0.8,4, 20, 100 nM for all cell lines (e.g. MCF-7, T47D, SKBR-3) except BT20for which the maximal dose is increased to 500 nM because saturation isnot achieved with 100 nM EGF.

[0140] 3. Aspirate culture media, transfer onto ice, and add lysisbuffer to lyse cells in situ.

[0141] 4. Scrape and transfer lysate to microfuge tube. Incubate on icefor 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min. (Centrifugationis optional.)

[0142] 5. Collect supernatants as lysates and aliquot for storage at−80° C. until use.

[0143] Assay:

[0144] Assay design: As illustrated diagrammatically in FIG. 4A,Her2-Her3 heterodimers (900) are quantified ratiometrically based on thebinding of cleaving probe (902) and binding compounds (904), (906), and(908). A photosensitizer indicated by “PS” is attached to cleaving probe(902) via an avidin-biotin linkage, and binding compounds (904), (906),and (908) are labeled with molecular tags Pro14, Pro10, and Pro11,respectively. Binding compound (904) is specific for a phosphorylationsite on Her3.

[0145] The total assay volume is 40 ul. The lysate volume is adjusted to30 ul with lysis buffer. The antibodies are diluted in lysis buffer upto 10 ul. Typically ˜5000 to 15000 cell-equivalent of lysates is usedper reaction. The detection limit is ˜1000 cell-equivalent of lysates.

[0146] Procedure: Final concentrations of pre-mixed binding compounds(i.e. molecular tag- or biotin-antibody conjugates) in reaction:

[0147] Pro4_anti-Her-2: 0.11 ug/ml

[0148] Pro10_anti-Her-1: 0.05-0.1 ug/ml

[0149] Pro11_anti-Her-3: 0.1 ug/ml

[0150] Pro2_anti-phospho-Tyr: 0.1 ug/ml

[0151] Biotin_anti-Her-2: 1-2 ug/ml

[0152] 1. To assay 96-well, add 10 ul antibody mix to 30 ul lysate andincubate for 1 hour at RT.

[0153] 2. Add 2 ul streptavidin-derivatized cleaving probe (final 2ug/well) to assay well and incubate for 45 min.

[0154] 3. Add 150 ul of PBS with 1% BSA to 96-well filter plate(Millipore MAGVN2250) and incubate for 1 hr at RT for blocking.

[0155] 4. Empty filter plate by vacuum suction. Transfer assay reactionsto filter plate and apply vacuum to empty.

[0156] 5. Add 200 ul wash buffer and apply vacuum to empty. Repeat onetime.

[0157] 6. Add 200 ul illumination buffer and apply vacuum to empty.Repeat one time.

[0158] 7. Add 30 ul illumination buffer and illuminate for 20 min.

[0159] 8. Transfer 10 ul of each reaction to CE assay plate for analysisusing an ABI3100 CE instrument with a 22 cm capillary (injectionconditions: 5 kV, 75 sec, 30° C.; run conditions: 600 sec, 30° C.).

[0160] Assay buffers are as follows:

[0161] Lysis Buffer (made fresh and stored on ice) Final ul Stock 1%Triton X-100 1000 10%  20 mM Tris-HCl (pH 7.5)  200   1 M 100 mM NaCl 200   5 M  50 mM NaF  500   1 M  50 mM Na beta-glycerophosphate 10000.5 M  1 mM Na₃VO₄  100 0.1 M  5 mM EDTA 100 0.5 M  10 ug/ml pepstatin 100   1 mg/ml 1 tablet (per 10 ml) Roche Complete protease N/A N/Ainhibitor (#1836170) Water 6500 N/A  10 ml Total Wash buffer (stored at4° C.) Final ml Stock 1% NP-40  50 10% 1x PBS  50 10x 150 mM NaCl  15  5 M  5 mM EDTA  5 0.5 M Water 380 N/A 500 ml Total Illuminationbuffer: Final ul Stock 0.005x PBS    50 1x CE std    3 100x 10 mMTris-HCl (pH 8.0) 0.1M 10 pM A160   1 nM 10 pM A315   1 nM 10 pM HABA  1 nM Water 10,000 ml N/A    10 ml Total

[0162] Data Analysis:

[0163] 1. Normalize relative fluorescence units (RFU) signal of eachmolecular tag against CE reference standard A315 (afluorescein-derivatized deoxyadenosine monophosphate that has known peakposition relative to molecular tags from the assay upon electrophoreticseparation).

[0164] 2. Subtract RFU of “no lysate” background control fromcorresponding molecular tag signals.

[0165] 3. Report heterodimerization for Her-1 or Her-3 as thecorresponding RFU ratiometric to RFU from Pro4_anti-Her-2 from assaywells using biotin-anti-Her-2.

[0166] 4. Report receptor phosphorylation for Her-1,2,3 as RFU fromPro2_PT100 anti-phospho-Tyr ratiometric to RFU from Pro4_anti-Her-2 fromassay wells using biotin-anti-Her-2.

[0167] Results of the assays are illustrated in FIGS. 4B-4H. FIG. 4Bshows the quantity of Her1-Her2 heterodimers increases on MCF-7 cellswith increasing concentrations of EGF, while the quantity of the samedimer show essentially no change with increasing concentrations of HRG.FIG. 4C shows the opposite result for Her2-Her3 heterodimers. That is,the quantity of Her2-Her3 heterodimers increases on MCF-7 cells withincreasing concentrations of HRG, while the quantity of the same dimershow essentially no change with increasing concentrations of EGF. FIGS.4D and 4E show the quantity of Her1-Her2 heterodimers increases onSKBR-3 cells and BT-20 cells, respectively, with increasingconcentrations of EGF.

Example 2 Analysis of Tissue Lysates for Her2 Heterodimerization andReceptor Phosphorylation

[0168] In this example, Her1-Her2 and Her2-Her3 heterodimers andphosphorylation states are measured in tissue lysates from human breastcancer specimens.

[0169] Sample Preparation:

[0170] 1. Snap frozen tissues are mechanically disrupted at the frozenstate by cutting.

[0171] 2. Transfer tissues to microfuge tube and add 3× tissue volumesof lysis buffer (from appendix I) followed by vortexing to dispersetissues in buffer.

[0172] 3. Incubate on ice for 30 min with intermittent vortexing to mix.

[0173] 4. Centrifuge at 14,000 rpm, 4° C., for 20 min.

[0174] 5. Collect supernatants as lysates and determine total proteinconcentration with BCA assay (Pierce) using a small aliquot.

[0175] 6. Aliquot the rest for storage at −80° C. until use.

[0176] Assay Design:

[0177] 1. The total assay volume is 40 ul.

[0178] 2. The lysates are tested in serial titration series of 40, 20,10, 5, 2.5, 1.25, 0.63, 0.31 ug total-equivalents and the volume isadjusted to 30 ul with lysis buffer. Data from the titration seriesconfirm the specificity of the dimerization or phosphorylation signals.

[0179] 3. A universal antibody mix comprising all eTag-antibodiesdiluted in lysis buffer is used at the following concentrations.

[0180] 4. Individual biotin-antibody for each receptor is addedseparately to the reactions.

[0181] 5. Three eTag assays are conducted with each tissue lysate, eachusing a different biotin-antibody corresponding to specific receptordimerization to be measured.

[0182] 6. Expression level of each receptor is determined from differentassay containing the biotin-antibody specific to the receptor.

[0183] 7. Dimerization and phosphorylation signals are determinedratiometrically only in the assay containing the biotin-anti-Her-2.

[0184] Assay controls: MCF-10A and MCF-7 cell lines are used asqualitative negative and positive controls, respectively. Cell lines areeither unstimulated or stimulated with 100 nM EGF or 100 nM HRG. Lysisbuffer is included as a background control when replacing the tissuesamples.

[0185] Final Concentrations of Pre-mixed Antibodies in Reactions:

[0186] Universal Antibody Mix:

[0187] Pro4_anti-Her-2: 0.1 ug/ml

[0188] Pro10_anti-Her-1: 0.05 ug/ml

[0189] Pro11_anti-Her-3: 0.1 ug/ml

[0190] Pro2_anti-phospho-Tyr: 0.01 ug/ml

[0191] Individual Biotin Antibody:

[0192] Biotin_anti-Her-1: 2 ug/ml

[0193] Biotin_anti-Her-2: 2 ug/ml

[0194] Biotin_anti-Her-3: 2 ug/ml

[0195] Procedure:

[0196] 1. Prepare antibody reaction mix by adding biotin antibody touniversal antibody mix.

[0197] 2. To assay 96-well, add 10 ul universal reaction mix to 30 ullysate and incubate for 1 hour at RT.

[0198] 3. Add 2 ul streptavidin-derivatized cleaving probe (final 2ug/well) to assay well and incubate for 45 min.

[0199] 4. Add 150 ul of PBS with 1% BSA to 96-well filter plate(Millipore MAGVN2250) and incubate for 1 hr at RT for blocking.

[0200] 5. Empty filter plate by vacuum suction. Transfer assay reactionsto filter plate and apply vacuum to empty.

[0201] 6. Add 200 ul wash buffer and apply vacuum to empty. Repeat onetime.

[0202] 7. Add 200 ul illumination buffer and apply vacuum to empty.Repeat one time.

[0203] 8. Add 30 ul illumination buffer and illuminate for 20 min.

[0204] 9. Transfer 10 ul of each reaction to CE assay plate for analysisusing ABI3100 capillary electrophoresis instrument with a 22 cmcapillary (injection conditions: 5 kV, 75 sec, 30° C.; run conditions:600 sec, 30° C.)

[0205] Data Analysis:

[0206] 1. Normalize RFU signal of each molecular tag against CEreference standard A315.

[0207] 2. Determine the cut-off values of RFU (each for dimerization orphosphorylation) below which ratios are not calculated because thesignals are too low to be reliable. Below the cut-off values, the RFUsignals are not titratable in the series of lysate dilution tested. Thevalues can be determined with a large set of normal tissues wheredimerization and phosphorylation signals are expected to be absent or atthe lowest. These values also represent the basal level of dimerizationor phosphorylation on the normal tissues to which tumor tissues will becompared.

[0208] 3. For the minority of normal tissues, if present, with RFUvalues above the cut-off, determine the individual RFU level andratiometric readouts of Her-1 or Her-3 heterodimerization orphosphorylation peaks detected. These samples represent outliers thatshould be used as matched donor controls for the corresponding tumortissue samples while scoring.

[0209] 4. For all tumor samples showing titratable RFU signals, use thelowest signal of each of Her-1, Her-2, Her-3, or phosphorylation fromthe tissue lysate titration series as the background. Subtract thisbackground from the molecular tag signals of the high dose lysates (e.g.40 ug) to yield the specific RFU signals. If there is no signal doseresponse in the titration series, all signals (which are usually verylow) are considered background and no specific signals can be used forratiometric analysis.

[0210] 5. Report heterodimerization for Her-1 or Her-3 as thecorresponding specific RFU ratiometric to the specific RFU fromPro4_anti-Her-2. If no specific RFU is obtained, the dimerization isnegative.

[0211] 6. Report receptor phosphorylation for Her-1,2,3 as specific RFUfrom Pro2_anti-phospho-Tyr ratiometric to the specific RFU fromPro4_anti-Her-2. If no specific RFU is obtained, the phosphorylation isnegative.

[0212] In FIGS. 5A-5C data shown are representative of multiplepatients' breast tissue samples tested with assays of the invention. Theclinical Her-2 status from immunohistochemistry (DAKO Herceptest) of 9out of 10 tumor samples was negative, indicative of either undetectableHer-2 staining, or staining of less than 10% of the tumor cells, or afaint and barely perceptible staining on part of the cell membrane ofmore than 10% tumor cells. The assays of the invention determined theexpression of Her-1, Her-2, and Her-3 on both normal and tumor tissues.The heterodimerization of Her1 and Her2 and of Her2 and Her3 wasdetected only in tumor tissues but not in any normal tissues.

Example 3 Analysis of Cell Lysates for Her1 or Her2 Homodimerization andReceptor Phosphorylation

[0213] Sample preparation was carried out essentially as described inExample 2. Her1 homodimerization was induced by treating the cell lineswith EGF or TGFα. For homodimerization of Her2 which does not have aligand, unstimulated SKBR-3 or MDA-MD-453 cells that overexpress Her2are compared to unstimulated MCF-7 cells that express a low level ofHer2.

[0214] Assay design: A monoclonal antibody specific to the receptor isseparately conjugated with either a molecular tag or biotin (that isthen linked to a photosensitizer via an avidin bridge), so that thecleaving probe and a binding compound compete to bind to the sameepitope in this example. Another binding compound is used that consistsof a second anibody recognizing an overlapping epitope on the receptor,so that a ratiometric signal can be generated as a measure ofhomodimerization. The signal derived from the second antibody alsoprovides a measure of the total amount of receptor in a sample. Thetotal amount of receptor is determined in a separate assay well.Receptor phosphorylation can be quantified together with eitherhomodimerization or total receptor amount.

[0215] Procedure: The assay volume is 40 ul and the general procedure issimilar to that of Example 2. Two assay wells, A and B, are set up foreach sample to quantify homodimerization and total amount of receptorseparately.

[0216] For Quantification of Her1-Her1 Homodimers:

[0217] Final Concentrations in Antibody Mix in Assay Well A:

[0218] Pro12_anti-Her-1: 0.05-0.1 ug/ml

[0219] Biotin_anti-Her-1: 1-2 ug/ml

[0220] Final Concentrations in Antibody Mix in Assay Well B:

[0221] Pro10_anti-Her-1: 0.05-0.1 ug/ml

[0222] Pro2_anti-phospho-Tyr: 0.1 ug/ml

[0223] Biotin_anti-Her-1: 1-2 ug/ml

[0224] For Quantification of Her2-Her2 Homodimers:

[0225] Final Concentrations in Antibody Mix in Assay Well A:

[0226] Pro4_anti-Her-1: 0.05-0.1 ug/ml

[0227] Biotin_anti-Her-1: 1-2 ug/ml

[0228] Final Concentrations in Antibody Mix in Assay Well B:

[0229] Pro4_anti-Her-1: 0.05-0.1 ug/ml

[0230] Pro2_anti-phospho-Tyr: 0.1 ug/ml

[0231] Biotin_anti-Her-1: 1-2 ug/ml

[0232] Data Analysis:

[0233] 1. Normalize RFU signal of each molecular tag against CEreference standard A315.

[0234] 2. Subtract RFU of “no lysate” background control fromcorresponding molecular tag signals.

[0235] 3. Report homodimerization for Her-1 or Her-2 as thecorresponding normalized RFU from assay well A as ratiometric tonormalized RFU of total receptor amount from the corresponding assaywell B.

[0236] 4. Report receptor phosphorylation for Her-1 or Her-2 homodimeras normalized RFU from Pro2_PT100 anti-phospho-Tyr from assay well B asratiometric to normalized RFU from total receptor amount from the sameassay well B.

[0237] Results of the assays are illustrated in FIGS. 6A-6B and FIG. 7.FIG. 6A shows that the quantity of Her1-Her1 homodimers on BT-20 cellsincreases with increasing concentration of EGF. FIG. 6B shows that thequantity of Her1 phosphorylation in BT-20 cells increases withincreasing EGF concentration. The detection of Her2-Her2 homodimers wasdemonstrated by comparison of signals from SKBR-3 cells expressing Her2with signals from MCF-7 cells that express reduced level of Her2 on thecell surface. As shown in the charts of FIG. 7, no specific titratableHer2-Her2 homodimer signals were detected with MCF-7 cells whereasHer2-Her2 homodimer signals from SKBR-3 cells were clearly above thesignals from MCF-7 cells

Example 4 Analysis of Cell Lysates for Her1-Her3 Heterodimerization andReceptor Phosphorylation

[0238] Samples are Prepared as Follows:

[0239] 1. Serum-starve breast cancer cell line culture overnight beforeuse.

[0240] 2. Stimulate cell lines with HRG in culture media for 10 minutesat 37° C. Exemplary doses of HRG are 0, 0.032, 0.16, 0.8, 4, 20, 100 nMfor T47D cells.

[0241] 3. Aspirate culture media, transfer onto ice, and add lysisbuffer to lyse cells in situ.

[0242] 4. Scrape and transfer lysate to microfuge tube. Incubate on icefor 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min. (Centrifugationis optional.)

[0243] 5. Collect supernatants as lysates and aliquot for storage at−80° C. until use.

[0244] Assay design: The total assay volume is 40 ul. The lysate volumeis adjusted to 30 ul with lysis buffer. The antibodies are diluted inlysis buffer up to 5 ul. Typically ˜5000 to 50000 cell-equivalent oflysates is used per reaction. Final concentrations of pre-mixedantibodies in reaction:

[0245] Pro10_anti-Her-1: 0.05-0.1 ug/ml

[0246] Pro11_anti-Her-3: 0.1 ug/ml

[0247] Pro2_anti-phospho-Tyr: 0.1 ug/ml

[0248] Biotin—anti-Her-3: 1-2 ug/ml

[0249] 1. To assay 96-well, add 5 ul antibody mix to 30 ul lysate andincubate for 1 hour at RT.

[0250] 2. Add 5 ul streptavidin-derivatized molecular scissor (final 4ug/well) to assay well and incubate for 45 min.

[0251] 3. Add 150 ul of PBS with 1% BSA to 96-well filter plate(Millipore MAGVN2250) and incubate for 1 hr at RT for blocking.

[0252] 4. Empty filter plate by vacuum suction. Transfer assay reactionsto filter plate and apply vacuum to empty.

[0253] 5. Add 200 ul wash buffer and apply vacuum to empty. Repeat onetime.

[0254] 6. Add 200 ul illumination buffer and apply vacuum to empty.Repeat one time.

[0255] 7. Add 30 ul illumination buffer and illuminate for 20 min.

[0256] 8. Transfer 10 ul of each reaction to CE assay plate for analysisusing ABI3100 capillary electrophoresis instrument with a 22 cmcapillary (injection conditions: 5 kV, 425 sec, 30° C.; run conditions:600 sec, 30° C.).

[0257] Data Analysis:

[0258] 1. Normalize RFU signal of each eTag reporter against CEreference standard A315.

[0259] 2. Subtract RFU of “no lysate” background control fromcorresponding eTag reporter signals.

[0260] 3. Report heterodimerization as the Her-1 derived Pro10 RFUratiometric to Pro11 RFU from anti-Her-3.

[0261] 4. Report receptor phosphorylation for Her-1/3 as RFU fromPro2_PT100 anti-phospho-Tyr ratiometric to RFU from Pro11_anti-Her-3from assay wells using biotin-anti-Her-3.

[0262] Results of the assay are illustrated in FIGS. 8A and 8B. The datashow that both Her1-Her3 heterodimerization and dimer phosphorylationincrease with increasing concentrations of HRG.

Example 5 Increase in Her1-Her3 Receptor Dimer Expression in Cancer CellLines in Response to Increase in Epidermal Growth Factor

[0263] In this example, Her1-Her3 heterodimers are measured in celllysates from cancer cell lines 22Rv1 and A549 after treatment withvarious concentrations of epidermal growth factor (EGF). Measurementsare made using three binding compounds and a cleaving probe as describedbelow.

[0264] Sample Preparation:

[0265] 1. Serum-starve breast cancer cell line culture overnight beforeuse.

[0266] 2. Stimulate cell lines with EGF in culture media for 10 minutesat 37° C. Exemplary doses of EGF applied to both cell lines variedbetween 0-100 nM.

[0267] 3. Aspirate culture media, transfer onto ice, and add lysisbuffer to lyse cells in situ.

[0268] 4. Scrape and transfer lysate to microfuge tube. Incubate on icefor 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min. (Centrifugationis optional.) Determine protein concentration.

[0269] 5. Collect supernatants as lysates and aliquot for storage at−80° C. until use.

[0270] The assay design is essentially the same as illustrated in FIG.4A, with the following exceptions: binding compounds (904), (906), and(908) are labeled with molecular tags Pro10, Pro11, and Pro2,respectively. The total assay volume is 40 ul. The lysate volume isadjusted to 30 ul with lysis buffer. The antibodies are diluted in lysisbuffer up to 5ul. Typically ˜5000 to 15000 cell-equivalent of lysates isused per reaction. The detection limit is ˜1000 cell-equivalent oflysates.

[0271] Procedure: Final concentrations of pre-mixed binding compounds(i.e. molecular tag- or biotin-antibody conjugates) in reaction:

[0272] Pro10_anti-Her-1: 0.05-0.1 ug/ml

[0273] Pro11_anti-Her-3: 0.1 ug/ml

[0274] Pro2_anti-phospho-Tyr: 0.1 to 0.2 ug/ml

[0275] Biotin_anti-Her-3: 1-2 ug/ml

[0276] 1. To assay 96-well, add 5 ul antibody mix to 30 ul lysate andincubate for 1 hour at RT.

[0277] 2. Add 5 ul streptavidin-derivatized cleaving probe (final 4ug/well) to assay well and incubate for 45 min.

[0278] 3. Add 150 ul of PBS with 1% BSA to 96-well filter plate(Millipore MAGVN2250) and incubate for 1 hr at RT for blocking.

[0279] 4. Empty filter plate by vacuum suction. Transfer assay reactionsto filter plate and apply vacuum to empty.

[0280] 5. Add 200 ul wash buffer and apply vacuum to empty. Repeat onetime.

[0281] 6. Add 200 ul illumination buffer and apply vacuum to empty.Repeat one time.

[0282] 7. Add 30 ul illumination buffer and illuminate for 20 min.

[0283] 8. Transfer 10 ul of each reaction to CE assay plate for analysisusing an ABI3100 CE instrument with a 22 cm capillary (injectionconditions: 5 kV, 70 sec, 30° C.; run conditions: 425 sec, 30° C.).

[0284] Assay buffers are as follows:

[0285] Lysis Buffer (made fresh and stored on ice) Final ul Stock 1%Triton X-100 1000 10%  20 mM Tris-HCl (pH 7.5)  500   1 M 100 mM NaCl 200   5 M  50 mM NaF  500   1 M  50 mM Na beta-glycerophosphate  5001.0 M  1 mM Na₃VO₄  100 0.1 M  5 mM EDTA  100 0.5 M  10 ug/ml pepstatin 100   1 mg/ml 1 tablet (per 10 ml) Roche Complete protease N/A N/Ainhibitor (#1836170) Water   7 ml N/A  10 ml Total Wash buffer (storedat 4° C.): 0.5% Triton X100 in 1x PBS. Illumination buffer: 0.005x PBS 50 1x CE std 1 (A27, ACLARA Biosciences, Inc.,   4 5000x Mountain View,CA) CE std 2 (fluorescein)   4 5000x Water 9942 N/A  10 ml Total

[0286] Data Analysis:

[0287] 1. Normalize relative fluorescence units (RFU) signal of eachmolecular tag against CE reference standard 2.

[0288] 2. Subtract RFU of “no lysate” background control fromcorresponding molecular tag signals.

[0289] 3. Report heterodimerization for Her-1 as the corresponding RFUratiometric to RFU from Pro11_anti-Her-3 from assay wells usingbiotin-anti-Her-3.

[0290] 4. Report receptor phosphorylation for Her-1,2,3 as RFU fromPro2_PT100 anti-phospho-Tyr ratiometric to RFU from Pro11_anti-Her-3from assay wells using biotin-anti-Her-3 (data not shown).

[0291]FIG. 9A and 9B show the increases in the numbers of Her1-Her3heterodimers on 22Rv1 and A549 cells, respectively, with increasingconcentrations of EGF.

Example 6 Occurrence of IGF-1R Heterodimers with Her1, Her2, and Her3 inBreast Tumor Tissue Lysates

[0292] In this example, cells from 12 different human breast tumortissues were assayed for the presence of Her1-IGF-1R, Her2-IGF-1R, andHer3-IGF-1R dimers using assays essentially the same as that illustratedin FIG. 4A. Sample Preparation was carried out as follows:

[0293] 1. Snap frozen tissues are mechanically disrupted at the frozenstate by cutting.

[0294] 2. Transfer tissues to microfuge tube and add 3× tissue volumesof lysis buffer followed by vortexing to disperse tissues in buffer.

[0295] 3. Incubate on ice for 30 min with intermittent vortexing to mix.

[0296] 4. Centrifuge at 14,000 rpm, 4° C., for 20 min.

[0297] 5. Collect supernatants as lysates and determine total proteinconcentration with BCA assay (Pierce) using a small aliquot.

[0298] 6. Aliquot the rest for storage at −80° C. until use.

[0299] The Assay was Set Up as Follows.

[0300] 1. The total assay volume is 40 ul.

[0301] 2. The lysates are tested in serial titration series of 40, 20,10, 5, 2.5, 1.25, 0.63, 0.31 ug total-equivalents and the volume isadjusted to 30 ul with lysis buffer. Data from the titration seriesconfirm the specificity of the dimerization.

[0302] 3. A universal antibody mix comprising of all binding compoundsand biotin antibody diluted in lysis buffer is used at concentrationsgiven below.

[0303] Final Concentrations of Pre-mixed Antibodies in Reactions:

[0304] Pro10_anti-Her-2: 0.1 ug/ml

[0305] Pro14_anti-Her-1: 0.1 ug/ml

[0306] Pro11_anti-Her-3: 0.1 ug/ml

[0307] Pro7_anti-IGF-1R: 0.1 ug/ml

[0308] Pro2_anti-phospho-Tyr: 0.2 ug/ml

[0309] Biotin_anti-Her-2: 2 ug/ml

[0310] Procedure:

[0311] 1. To assay 96-wells, add 5 ul universal reaction mix to 30 ullysate and incubate for I hour at RT.

[0312] 2. Add 5 ul strepatvidin-derivatized molecular scissor, i.e.cleaving probe (final 4 ug/well) to assay well and incubate for 45 min.

[0313] 3. Add 150 ul of of PBS with 1% BSA to 96-well filter plate(Millipore MAGVN2250) and incubate for 1 hr at RT for blocking.

[0314] 4. Empty filter plate by vacuum suction. Transfer assay reactionsto filter plate and apply vacuum to empty.

[0315] 5. Add 200 ul wash buffer and apply vacuum to empty. Repeat onetime.

[0316] 6. Add 200 ul illumination buffer and apply vacuum to empty.Repeat one time.

[0317] 7. Add 30 ul illumination buffer and illuminate for 20 min.

[0318] 8. Transfer 10 ul of each reaction to CE assay plate for analysisusing: (i) CE equipment: ABI3100, 22 cm capillary, (ii) CE injectionconditions: 5 kV, 70 sec, 30° C., and (iii) CE run conditions: 425 sec,30° C.

[0319] Data Analysis:

[0320] 1. Normalize RFU signal of each molecular tag against CEreference standard 1.

[0321] 2. Look for titratable signals for each molecular tag. Signalsthat do not titrate are assumed to be non-specific signals and are notused for data interpretation. A cut off value is determined based on thevalues from a large set of normal tissues where dimerization signals areexpected to be absent or at the lowest. These values also represent thebasal level of dimerization on the normal tissues to which tumor tissuesare compared.

[0322] 3. Heterodimerization is reported for IGF-1R with Her-1 or Her-2or Her-3 as the corresponding specific RFU.

[0323] Two out of the twelve breast tumors assayed expressedHer1-IGF-1R, Her2-IGF-1R, and Her3-IGF-1R heterodimers, as shown inFIGS. 10A-C. The lines in each figure panel shows the trend betweenreceptor heterodimer quantity measured and amount of lysate assayed forthe two breast tumor samples that were positive for the indicatedheterodimers.

Example 7 PI3K/Her-3 Receptor Activation Complex

[0324] In this example, assays were designed as shown in FIGS. 11A and11C to measure a receptor complex comprising Her2, Her3, and PI3K inbreast cancer cell line, MCF-7. Binding compound (1106) having a firstmolecular tag (“mT₁” in the figure and “eTag1” below) is specific forthe extracellular domain of Her3 receptor (1102), binding compound(1110) having a second molecular tag (“mT2” in the figure and “eTag2”below) is specific for the p185 component (1111) of PI3K protein (1100),and cleaving probe (1108) having a photosensitizer attached (is specificfor the intracellular domain of Her3 receptor (1102) where “H2”indicates a Her2 receptor (1104), “H3” indicates a Her3 receptor (1102),“p85” and “p110” are components of PI3 kinase (1100), which binds to aphosphorylation site of H3 (denoted by “P”) through its p85 moiety. Thetwo assay designs are similar, except that in the design of FIG. 11A thecleaving probe is specific for the Her3 receptor, and in the design ofFIG. 11C, the cleaving probe is specific for the p85 component of PI3kinase. The assays were carried out as follows.

[0325] Sample Preparation:

[0326] 1. Serum-starve breast cancer cell line culture overnight beforeuse.

[0327] 2. Stimulate cell lines with HRG in culture media for 10 minutesat 37° C. Exemplary doses of HRG are 0, 0.032, 0.16, 0.8, 4, 20, 100 nMfor MCF-7 cells.

[0328] 3. Aspirate culture media, transfer onto ice, and add lysisbuffer (described above to lyse cells in situ.

[0329] 4. Scrape and transfer lysate to microfuge tube. Incubate on icefor 30 min. Microfuge at 14,000 rpm, 4° C., for 10 min.

[0330] 5. Collect supernatants as lysates and aliquot for storage at−80° C. until use. Lysis Buffer (made fresh and stored on ice): Final ulStock 1% Triton X-100 1000 10% 20 mM Tris-HCl (pH 7.5)  200   1 M 100 mMNaCl  200   5 M 50 mM NaF  500   1 M 50 mM Na beta-glycerophosphate 10000.5 M 1 mM Na₃VO₄  100 0.1 M 5 mM EDTA  100 0.5 M 10 ug/ml pepstatin 100   1 mg/ml 1 tablet (per 10 ml) Roche Complete protease N/A N/Ainhibitor (#1836170) Water 6500 N/A  10 ml Total

[0331] Assay design: Receptor complex formation is quantifiedratiometrically based on the schematics illustrated in each figure. Thatis, the readout of the assays are the peak ratios of molecular tags,eTag2/eTag1.

[0332] The total assay volume is 40 ul. The lysate volume is adjusted to10 ul with lysis buffer. The antibodies are diluted in lysis buffer upto 20 ul. Typically ˜5000 to 500,000 cell-equivalent of lysates is usedper reaction.

[0333] Procedure: Working concentrations of pre-mixed antibodies priorto adding into reaction: For Her-3/PI3K complex with cleaving probe atHer-3 (the design of FIG. 11A)

[0334] eTag1_anti-Her-3 at 10 nM (eTag1 was Pro14 in this assay)

[0335] eTag2_anti-PI3K at 10 nM (eTag2 was Pro1 in this assay)

[0336] Biotin_anti-Her-3 at 20 nM

[0337] Universal Standard US-1 at 700 nM

[0338] [The Universal Standard US-1 is BSA conjugated with biotin andmolecular tag Pro8, which is used to normalize the amount ofstreptavidin-photosensitizer beads in an assay]. The molecular tags wereattached directly to antibodies by reacting an NHS-ester of a moleculartag precursor with free amines on the antibodies using conventionaltechniques, e.g. Hermanson (cited above).

[0339] For Her-3/PI3K Complex with Cleaving Probe at PI3K (the Design ofFIG. 11C):

[0340] eTag1_anti-PI3K at 10 nM (eTag1 was Pro1 in this assay)

[0341] eTag2_anti-Her-3 at 10 nM (eTag2 was Pro14 in this assay)

[0342] Biotin_anti-PI3K at 20 nM

[0343] Universal Standard US-1 at 700 nM

[0344] 9. To assay 96-well filter plate (Millipore MAGVN2250), add 20 ulantibody mix to 10 ul lysate and incubate for 1 hour at 4° C.

[0345] 10. Add 10 ul streptavidin-derivatized cleaving probe (final 4ug/well) to assay well and incubate for 40 min.

[0346] 11. Add 200 ul wash buffer and apply vacuum to empty.

[0347] 12. Add 30 ul illumination buffer and illuminate.

[0348] 13. Transfer 10 ul of each reaction to CE assay plate foranalysis.

[0349] Data Analysis:

[0350] 1. Normalize relative fluorescence units (RFU) signal of eachmolecular tag against that of internal Universal Standard US-1.

[0351] 2. Subtract RFU of “no lysate” background control fromcorresponding normalized eTag reporter signals.

[0352] 3. Report receptor complex formation as the ratiometric ofnormalized eTag2/eTag1 signal (shown in FIGS. 11B and 11D).

Example 8 Shc/Her-3 Receptor-Adaptor Interaction

[0353] In this example, an assays were designed as shown in FIGS. 12Aand 12C. In FIG. 12A, Her2 receptor (1200) and Her3 receptor (1202) forma dimer in cell surface membrane (1204) and each receptor is representedas having phosphorylated sites (1209 and 1210). Shc proteins (1206 and1208) bind to phosphylation sites (1210) and (1209), respectively. Afirst binding compound (1214) and cleaving probe (1216) are specific fordifferent antigenic determinants of the extracellular domain of Her2receptor (1200). A second binding compound (1212) is specific for Shcproteins (1206 and 1208). The assay designs of FIGS. 12A and 12C aresimilar, except that in the design of FIG. 12A the cleaving probe isspecific for the Her2 receptor, and in the design of FIG. 12C, thecleaving probe is specific for the Her3 receptor. Thus, in the formercase, total Her2 receptor is measured, whereas in the latter case totalHer3 receptor is measured. The assays were carried out as follows.Sample preparation was carried out as above (Example 7).

[0354] Assay design: Receptor complex formation is quantifiedratiometrically based on the schematics illustrated in each figure. Thatis, in FIGS. 12B and 12D the readout of the assays are the peak ratiosof mT₂/mT₁ as a function of HRG concentration.

[0355] The total assay volume is 40 ul. The lysate volume is adjusted to10 ul with lysis buffer. The antibodies are diluted in lysis buffer upto 20 ul. Typically about 5000 to 500,000 cell-equivalent of lysates isused per reaction.

[0356] Procedure: Working Concentrations of Pre-mixed Antibodies Priorto Adding into Reaction:

[0357] For Her-3/Shc Complex with Cleaving Probe at Her-3 (the Design ofFIG. 12B):

[0358] eTag1_anti-Her-3 at 10 nM (eTag1 was Pro14 in this assay)

[0359] eTag2_anti-Shc at 10 nM (eTag2 was Pro12 in this assay)

[0360] eTag3_anti-phospho-Tyr at 10 nM (eTag3 was Pro2 in this assay)

[0361] Biotin_anti-Her-3 at 20 nM

[0362] Universal Standard US-1 at 700 nM

[0363] For Her-2/Shc Complex with Cleaving Probe at Her-2 (the Design of12A):

[0364] eTag1_anti-Her-2 at 10 nM (eTag1 was Pro14 in this assay)

[0365] eTag2_anti-Shc at 10 nM (eTag2 was Pro12 in this assay)

[0366] eTag3_anti-phospho-Tyr at 10 nM (eTag3 was Pro2 in this assay)

[0367] Biotin_anti-Her-2 at 20 nM

[0368] Universal Standard US-1 at 700 nM

[0369] 1. To assay 96-well filter plate (Millipore MAGVN2250), add 20 ulantibody mix to 10 ul lysate and incubate for 1 hour at 4° C.

[0370] 2. Add 10 ul streptavidin-derivatized cleaving probe (final 4ug/well) to assay well and incubate for 40 min.

[0371] 3. Add 200 ul wash buffer and apply vacuum to empty.

[0372] 4. Add 30 ul illumination buffer and illuminate.

[0373] 5. Transfer 10 ul of each reaction to CE assay plate foranalysis.

[0374] Data Analysis:

[0375] 1. Normalize relative fluorescence units (RFU) signal of eachmolecular tag against that of internal Universal Standard US-1.

[0376] 2. Subtract RFU of “no lysate” background control fromcorresponding normalized signals for molecular tags.

[0377] 3. Report receptor complex formation as the ratiometric ofnormalized mT₂/mT₁ signals (shown in FIGS. 12B and 12D) and receptorphosphorylation (data not shown) as mT3/mT1 signals.

Example 9 Correlation Between Her2-Her3 Heterodimer Measurements andHer3-PI3K Complex Measurements in Breast Tumor Samples

[0378] In this example, human breast tumor samples were separatelyassayed using the methods described above to determine the amounts ofHer2-Her3 heterodimers and the amounts of Her3-PI3K complex. FIG. 13illustrates data obtained from such assays, which shows that the twomeasurements are correlated.

Example 10 Expression of Her1-Her2 and Her2-Her3 Heterodimers in BreastTumor Tissue Lysates and Normal Tissue Lysates

[0379] Frozen human breast tumor tissue samples and normal tissuesamples were obtained from the William Bainbridge Genomic Foundation(Bainbridge Island, Wash.). Assays having a format as shown in FIG. 3Ewere performed on 32 tumor tissue samples and 30 normal tissue samples.Tumor tissues consisted of a mixture of tumor and normal cells thatvaried from about 25 percent to over 90 percent according to pathologydata supplied with the tissues by the vendor. Samples were prepared andthe assays carried out essentially as described for Examples 2 and 6.Data is reported as peak area or intensity of the separated moleculartag released from the binding compound specifically bound to thereceptor opposite the cleaving probe, i.e. the molecular tagcorresponding to “mT₁” in FIG. 3E. No attempt was made to normalize thesignals generated according to percentage tumor cells in a sample.

[0380] The data from these measurements are shown in FIG. 14A (Her1-Her2heterodimer measurements) and FIG. 14B (Her2-Her3 heterodimermeasurements), where the open squares (□) indicate measurements on tumortissues and the solid diamonds (♦) indicate measurements on normaltissues. The data show that tumor cells in substantial fractions of thetumor tissue samples express large amounts of Her1-Her2 heterodimers andHer2-Her3 heterodimers relative to those expressed in the cells of thenormal tissue samples.

Example 11 Measurement of Receptor Dimers in Formalin Fixed ParaffinEmbedded Tissue Samples

[0381] In this example, model fixed tissues made from pelleted celllines were assayed for the presence of Her receptor dimers. The assaydesign for heterodimers was essentially the same as that described inFIG. 4A, with exceptions as noted below. That is, four components areemployed: (i) a cleaving probe comprising a biotinylated monoclonalantibody conjugated to a cleavage-inducing moiety (in this example, aphotosensitizer-derivatized streptavidin, as illustrated in FIG. 3E) andspecific for one of the receptors of the dimer, (ii) a monoclonalantibody derivatized with a first molecular tag and specific for thesame receptor as the cleaving probe, (iii) a monoclonal antibodyderivatized with a second molecular tag and specific for the receptoropposite to that the cleaving probe is specific for, and (iv) amonoclonal antibody derivatized with a third molecular tag and specificfor an intracellular phosphorylated tyrosine. The assay design forhomodimers was essentially the same as that described in FIG. 1D, withexceptions as noted below.

[0382] In each case, model fixed tissues were prepared as follows: cellsgrown on tissue culture plates were stimulated with either EGF or HRG asdescribed in the prior examples, after which they were washed andremoved by scrapping. The removed cells were centrifuged to form apellet, after which formalin was added and the mixture was incubatedovernight at 4° C. The fixed pellet was embedded in paraffin using aMiles Tissue Tek III Embedding Center, after which 10 μm tissue sectionswere sliced from the pellet using a microtome (Leica model 2145). Tissuesections were placed on positively charged glass microscope slides(usually multiple tissue sections per slide) and baked for 1 hr at 60°C.

[0383] Tissue sections on the slides were assayed as follows: Tissuesections on a slide were de-waxed with EZ-Dewax reagent (Biogenex, SanRamon, Calif.) using the manufacturer's recommended protocol. Briefly,500 μL EZ-Dewax was added to each tissue section and the sections wereincubated at RT for 5 min, after which the slide was washed with 70%EtOH. This step was repeated and the slide was finally rinsed withdeionized water, after which the slide was incubated in water at RT for20 min. The slide was then immersed into a 1X Antigen Retrieval solution(Biogenesis, Brentwood, N.H.) at pH 10, after which it was heated for 15min in a microwave oven (5 min at high power setting followed by 10 minat a low power setting). After cooling to RT (about 45 min), the slidewas placed in a water bath for 5 min, then dried. Tissue sections on thedried slide were circled with a hydrophobic wax pen to create regionscapable of containing reagents placed on the tissue sections (asillustrated in FIGS. 1H-1I), after which the slide was washed threetimes in 1X Perm/Wash (BD Biosciences). To each section 50-100 μLblocking buffer was added, and the slide was placed in a coveredhumidified box containing deionized water for 2 hr at 4° C, after whichthe blocking buffer was removed from each section by suction. (Blockingbuffer is 1X Perm/Wash solution with protease inhibitors (Roche),phosphatase inhibitors (sodium floride, sodium vanadate, β-glycerolphosphate), and 10% mouse serum). To each section 40-50 μL of antibodymix containing binding compounds and cleaving probe was added (each at 5μg/mL, except that biotin-Ab5 (anti-Her1 ) was at 10 μg/mL in theHer1-Her2 assay), and the slide was placed in a humidified box overnightat 4° C. The sections were then washed three times with 100 μL Perm/Washcontaining protease and phosphatase inhibitors, after which 50 μL ofphotosensitizer in 1X Perm/Wash solution (containing protease andphosphatase inhibitors) was added. The slide was then incubated for1-1.5 hr at 4° C. in the dark in a humidified box, after which thephotosensitizer was removed by suction while keeping the slide in thedark. While remaining in the dark, the slide was then immersed in 0.01XPBS and incubated on ice for 1 hr. The slide was remove from the PBS,dried, and to each section, 40-50 μL 0.01×PBS with 2 pM fluorescein wasadded, after which it was illuminated with a high power laser diode(GaAIAs IR emitter, model OD-880W, OPTO DIODE CORP, Newbury Park,Calif.) for 1 hr. The fluorescein acts as a standard to assist incorrelating peaks in an electropherogram with moleuclar tags. Afterillumination, the solution covering each tissue section was mixed bygentle pipeting and transferred to a CE plate for analysis on an AppliedBiosystems (Foster City, Calif.) model 3100 capillary electrophoresisinstrument.

[0384]FIG. 15A shows data from analysis of Her1-Her1 homodimers andreceptor phosphorylation in sections from fixed pellets of breastadenocarcinoma cell line, MDA-MB468 (ATCC accession no. HTB-132),prepared from either non-stimulated cells or cells stimulated with 100nM EGF. Biotinylated anti-Her1 monoclonal antibody (Labvision) at 2μg/mL was use as the primary antibody of the cleaving probe (forcleavage methylene-blue derivatized streptavidin (described above) wasattached through the biotin). Pro10-derivatized anti-Her1 monoclonalantibody (Labvision) at 2 μg/mL was used to measure homodimerized Her1.Pro1-derivatized anti-Her1 monoclonal antibody (Labvision) at 0.8 μg/mLwas used to measure total Her1. Unlabeled antibody Ab-5 was alsoincluded in the reactions at 3.2 μg/mL. Pro2-derivatized monoclonalantibody (anti-phosphorylated-Tyr, Cell Signaling) at 0.5 μg/mL was usedto measure intracellular phosphorylation. The data from fixed tissuemeasurements confirm and are consistent with measurements on celllysates that show increases in Her1 -Her1 homodimer expression andintracellular phosphoryation due to EGF stimulation.

[0385]FIG. 15B shows data from analysis of Her2-Her2 homodimers andreceptor phosphorylation in sections from fixed pellets of breast cancercell lines MCF-7 and SKBR-3. All monoclonal antibodies used as cleavingprobes or binding compounds were used at concentrations of 5 μg/mL. Inorder to generate better cleavage, in this assay two cleaving probeswere employed, one directed to an extracellular antigenic determinant ofHer2 and one directed to an intracellular antigenic determinant of Her2.The data from fixed tissue measurements confirm that SKBR3 cells expresshigher levels of Her2-Her2 homodimers than MCF-7 cells.

[0386]FIG. 15C shows data from analysis of Her1-Her2 heterodimers andreceptor phosphorylation in sections from fixed pellets of breastadenocarcinoma cell line, MCF-7, prepared from either non-stimulatedcells or cells stimulated with 40 nM EGF. Two cleaving probes wereemployed one comprising anti-Her1 monoclonal antibody (at 5 μg/mL) andthe other comprising anti-Her1 monoclonal antibody (at 10 μg/mL) (bothfrom Labvision) in order to increase the rate at which molecular tagswere released. The data show that increases in Her1-Her2 heterodimerexpression due to EGF stimulation is detected in fixed tissue.

[0387]FIG. 15D shows data from analysis of Her1-Her2 heterodimers andreceptor phosphorylation in sections from fixed pellets of breastadenocarcinoma cell line, 22Rv1, prepared from either non-stimulatedcells or cells stimulated with 100 nM EGF. Again, measurements on fixedtissues demonstrates the up-regulation of Her1-Her2 dimers and Herreceptor phosphorylation in response to treatment with EGF.

[0388]FIG. 15E shows data from analysis of Her2-Her3 heterodimers andreceptor phosphorylation in sections from fixed pellets of breastadenocarcinoma cell line, MCF-7, prepared from either non-stimulatedcells or cells stimulated with 40 nM HRG. In this example, bindingreactions and cleavage reactions took place in tubes containingsections, rather than microscope slides. Otherwise, the protocol wasessentially the same as that for detecting the Her1-Her2 dimers. Thedata show that increases in Her2-Her3 heterodimer expression due to HRGstimulation is detected in fixed tissue.

[0389]FIG. 15F shows data from analysis of Her2-Her3 heterodimers andPI3K-Her3 dimers in sections from fixed pellets of MCF-7 cells eithernon-stimulated or stimulated with 40 nM HRG. The assay design forPI3K-Her3 was essentially as described in FIG. 11A. The above fixationprotocol was followed in both cases, except that neither sample wastreated with antigen retrieval reagents. The data show that Her2-Her3dimers increased with treatment by HRG, but that the amount of PI3K-Her3dimer remained essentially unchanged.

[0390]FIG. 15G shows data from analysis of total PI3K, total Her2-Her3dimer, and total Her3 all relative to amount of tubulin. Tubulin wasmeasured in a conventional sandwich-type assay employing a cleavageprobe and a binding compound with a molecular tag. Tubulin was measuredto test procedures for normalizing dimer measurement against a targetrepresentative of total cell number in a sample, which may be requiredfor measurements on samples with heterogeneous cell types. The data showthat the ratios of PI3K-Her3 and Her2-Her3 to tubulin are qualitativelythe same as the measurements directly on PI3K-Her3 and Her2-Her3.

What is claimed is:
 1. A method of determining disease status of apatient suffering from a disease characterized by aberrant expression ofone or more ErbB cell surface receptor complexes, the method comprisingthe steps of: measuring directly in a patient sample an amount of eachof one or more ErbB cell surface receptor complexes; comparing each suchamount to its corresponding amount in a reference sample; andcorrelating differences in the amounts from the patient sample and therespective corresponding amounts from the reference sample to thedisease status the patient.
 2. The method of claim 1 wherein saiddisease is a cancer and wherein said patient sample is a fixed tissuesample, a frozen tissue sample, or circulating epithelial cells.
 3. Themethod of claim 2 wherein said one or more ErbB cell surface receptorcomplexes are selected from the group consisting of Her1-Her1homodimers, Her2-Her2 homodimers, Her1-Her3 receptor dimers, Her2-Her4receptor dimers, Her1-PI3K complexes, Her2-PI3K complexes, Her3-PI3Kcomplexes, Her1-SHC complexes, Her2-SHC complexes, Her3-SHC complexes,Her1-IGF-1R receptor dimers, Her2-IGF-1R receptor dimers, Her3-IGF-1Rreceptor dimers, Her1-PDGFR receptor dimers, Her2-PDGFR receptor dimers,Her3-PDGFR receptor dimers, p95Her2-Her3 receptor dimers, p95Her2-Her2receptor dimers, p95Her2-Her1 receptor dimers, EGFRvIII-Her1 receptordimers, EGFRvIII-Her2 receptor dimers, and EGFRvIII-Her3 receptordimers.
 4. The method of claim 3 wherein each of said one or more ErbBcell surface receptor complexes are determined by the steps of:providing for each of said one or more Her complexes a reagent paircomprising a cleaving probe having a cleavage-inducing moiety with aneffective proximity, and one or more binding compounds each having oneor more molecular tags attached thereto by a cleavable linkage, themolecular tags of different binding compounds having differentseparation characteristics; mixing the cleaving probe and the one ormore binding compounds for each of said one or more Her complexes withsaid patient sample such that the cleaving probe and the one or morebinding compounds specifically bind to their respective Her complexesand the cleavable linkages of the one or more binding compounds arewithin the effective proximity of the cleavage-inducing moiety so thatmolecular tags are released; and separating and identifying the releasedmolecular tags to determine the presence or absence or the amount ofsaid one or more ErbB cell surface receptor complexes in said patientsample.
 5. The method of claim 4 wherein said patient sample is saidfixed tissue sample or said frozen tissue sample.
 6. The methodaccording to claims 3, 4, or 5 wherein said disease status isresponsiveness of said patient to treatment with a dimer-acting drug. 7.The method of claim 6 wherein said cancer is selected from the groupconsisting of breast cancer, ovarian cancer, prostate cancer, andcolorectal cancer.
 8. The method of claim 1 wherein said one or moreErbB cell surface receptor complexes are one or more heterodimers with aPDGF receptor.
 9. The method of claim 8 wherein said one or moreheterodimers are selected from the group consisting of Her1-PDGFRreceptor dimers, Her2-PDGFR receptor dimers, and Her3-PDGFR receptordimers.
 10. The method of claim 9 wherein said patient sample is saidfixed tissue sample or said frozen tissue sample.
 11. The method ofclaim 10 wherein said one or more heterodimers are determined by thesteps of: providing for each of said one or more heterodimers a reagentpair comprising a cleaving probe having a cleavage-inducing moiety withan effective proximity, and one or more binding compounds each havingone or more molecular tags attached thereto by a cleavable linkage, themolecular tags of different binding compounds having differentseparation characteristics; mixing the cleaving probe and the one ormore binding compounds for each of said one or more heterodimers withsaid patient sample such that the cleaving probe and the one or morebinding compounds specifically bind to their respective heterodimers andthe cleavable linkages of the one or more binding compounds are withinthe effective proximity of the cleavage-inducing moiety so thatmolecular tags are released; and separating and identifying the releasedmolecular tags to determine the presence or absence or the amount ofsaid one or more heterodimers in said patient sample.
 12. The methodaccording to claims 8, 9, 10, or 11 wherein said disease is cancer orwherein said disease is associated with an aberrant fibrotic condition.13. The method of claim 12 wherein said cancer is selected from thegroup consisting of breast cancer, ovarian cancer, and glioblastoma. 14.The method of claim 1 wherein said patient sample is a fixed tissuesample and wherein said disease is cancer and wherein said one or moreErbB cell surface receptor complexes are Her receptor dimers selectedfrom the group consisting of Her1-Her1, Her1-Her3, Her1-Her4, Her2-Her2,Her3-Her4, and Her4-Her4.
 15. The method of claim 14 wherein said one ormore Her receptor dimers are determined by the steps of: providing foreach of said one or more Her receptor dimers a reagent pair comprising acleaving probe having a cleavage-inducing moiety with an effectiveproximity, and one or more binding compounds each having one or moremolecular tags attached thereto by a cleavable linkage, the moleculartags of different binding compounds having different separationcharacteristics; mixing the cleaving probe and the one or more bindingcompounds for each of said one or more Her receptor dimers with saidpatient sample such that the cleaving probe and the one or more bindingcompounds specifically bind to their respective Her receptor dimers andthe cleavable linkages of the one or more binding compounds are withinthe effective proximity of the cleavage-inducing moiety so thatmolecular tags are released; and separating and identifying the releasedmolecular tags to determine the presence or absence or the amount ofsaid one or more Her receptor dimers in said fixed tissue sample.
 16. Amethod of selecting a patient for treatment of a cancer with one or moreErbB-dimer-acting drugs, the method comprising the steps of: isolating apatient sample containing cancer cells from a patient; measuringdirectly in the patient sample an amount of each of one or more ErbBcell surface receptor dimers; comparing each such amount to itscorresponding amount from a reference sample; and selecting the patientfor treatment with one or more ErbB dimer-acting drugs whenever anamount of one or more cell surface receptor dimers from the patientsample exceeds the respective corresponding amount from the referencesample.
 17. The method of claim 16 wherein said patient sample is afixed tissue sample, a frozen tissue sample, or circulating epithelialcells.
 18. The method of claim 17 wherein said ErbB cell surfacereceptor dimer contains a Her1 receptor and said dimer-acting drug isselected from the group consisting of Cetuximab (Erbitux), Trastuzumab(Herceptin), h-R3 (TheraCIM), ABX-EGF, MDX447, ZD-1839 (Iressa), OSI-774(Tarceva), PKI 166, GW572016, CI-1033, EKB-569, and EMD
 72000. 19. Themethod of claim 18 wherein said ErbB cell surface receptor dimer isselected from the group consisting of Her1-Her1, Her1-Her2, Her1-Her3,Her1-Her4.
 20. The method of claim 19 wherein said ErbB cell surfacereceptor dimer is selected from the group consisting of Her1-Her1,Her1-Her3, Her1-Her4.
 21. The method of claim 20 wherein said ErbB cellsurface receptor dimer is Her1-Her1.
 22. The method of claim 21 whereinsaid patient sample is a fixed tissue sample and wherein said ErbBdimer-acting drug selected from the group consisting of Cetuximab(Erbitux), ABX-EGF, MDX-447, ZD-1839 (Iressa), OSI-774 (Tarceva), PKI166, GW572016, CI-1033, EKB-569, and EMD
 72000. 23. The method accordingto claims 16, 17, 18, 19, 20, 21, or 22 wherein said one or more ErbBcell surface receptor dimers are determined by the steps of: providingfor each of said one or more ErbB cell surface receptor dimers a reagentpair comprising a cleaving probe having a cleavage-inducing moiety withan effective proximity, and one or more binding compounds each havingone or more molecular tags attached thereto by a cleavable linkage, themolecular tags of different binding compounds having differentseparation characteristics; mixing the cleaving probe and the one ormore binding compounds for each of said one or more ErbB cell surfacereceptor dimers with said patient sample such that the cleaving probeand the one or more binding compounds specifically bind to theirrespective ErbB cell surface receptor dimers and the cleavable linkagesof the one or more binding compounds are within the effective proximityof the cleavage-inducing moiety so that molecular tags are released; andseparating and identifying the released molecular tags to determine thepresence or absence or the amount of said one or more ErbB cell surfacereceptor dimers in said fixed tissue sample.
 24. A method of determininga cancer status of a patient suffering from a cancer characterized byaberrant expression of one or more ErbB cell surface receptor complexes,the method comprising the steps of: measuring directly in a patientsample an amount of each of one or more ErbB cell surface receptorcomplexes; comparing each such amount to its corresponding amount in areference sample; and correlating differences in the amounts from thepatient sample and the respective corresponding amounts from thereference sample to the disease status the patient; wherein said one ormore ErbB cell surface receptor complexes are selected from the groupconsisting of Her1-PI3K complexes, Her2-PI3K complexes, Her3-PI3Kcomplexes, Her1-SHC complexes, Her2-SHC complexes, Her3-SHC complexes,Her1-IGF-1R receptor dimers, Her2-IGF-1R receptor dimers, Her3-IGF-1Rreceptor dimers, Her1-PDGFR receptor dimers, Her2-PDGFR receptor dimers,Her3-PDGFR receptor dimers, p95Her2-Her3 receptor dimers, p95Her2-Her2receptor dimers, p95Her2-Her1 receptor dimers, EGFRvIII-Her1 receptordimers, EGFRvIII-Her2 receptor dimers, and EGFRvIII-Her3 receptordimers.
 25. The method of claim 24 wherein said disease is a cancer andwherein said patient sample is a fixed tissue sample, a frozen tissuesample, or circulating epithelial cells.
 26. The method of claim 25wherein each of said one or more ErbB cell surface receptor complexesare determined by the steps of: providing for each of said one or moreErbB cell surface receptor complexes a reagent pair comprising acleaving probe having a cleavage-inducing moiety with an effectiveproximity, and one or more binding compounds each having one or moremolecular tags attached thereto by a cleavable linkage, the moleculartags of different binding compounds having different separationcharacteristics; mixing the cleaving probe and the one or more bindingcompounds for each of said one or more ErbB cell surface receptorcomplexes with said patient sample such that the cleaving probe and theone or more binding compounds specifically bind to their respective ErbBcell surface receptor complexes and the cleavable linkages of the one ormore binding compounds are within the effective proximity of thecleavage-inducing moiety so that molecular tags are released; andseparating and identifying the released molecular tags to determine thepresence or absence or the amount of said one or more ErbB cell surfacereceptor complexes in said patient sample.
 27. The method of claim 26wherein said patient sample is said fixed tissue sample or said frozentissue sample.
 28. The method according to claims 24, 25, 26, or 27wherein said cancer status is responsiveness of said patient totreatment with a dimer-acting drug.
 29. The method of claim 28 whereinsaid cancer is selected from the group consisting of breast cancer,ovarian cancer, prostate cancer, and colorectal cancer.
 30. A method ofdetermining disease status of a patient suffering from a diseasecharacterized by aberrant expression of one or more ErbB cell surfacereceptor complexes, the method comprising the steps of: measuringdirectly in a patient sample an amount of each of one or more ErbB cellsurface receptor complexes; comparing each such amount to itscorresponding amount in a reference sample; correlating differences inthe amounts from the patient sample and the respective correspondingamounts from the reference sample to the disease status the patient; andwherein each of said one or more ErbB cell surface receptor complexesare determined by the steps of: providing for each of said one or moreErbB cell surface receptor complexes and one or more tissue indicators areagent pair comprising a cleaving probe having a cleavage-inducingmoiety with an effective proximity, and one or more binding compoundseach having one or more molecular tags attached thereto by a cleavablelinkage, the molecular tags of different binding compounds havingdifferent separation characteristics; mixing the cleaving probe and theone or more binding compounds for each of said one or more ErbB cellsurface receptor complexes and one or more tissue indicators with saidpatient sample such that the cleaving probe and the one or more bindingcompounds specifically bind to their respective targets and thecleavable linkages of the one or more binding compounds are within theeffective proximity of the cleavage-inducing moiety so that moleculartags are released; and separating and identifying the released moleculartags to determine the presence or absence or the amount of said one ormore ErbB cell surface receptor complexes in said patient sample. 31.The method of claim 30 wherein said disease is a cancer and wherein saidpatient sample is a fixed tissue sample, a frozen tissue sample, orcirculating epithelial cells.
 32. The method of claim 31 wherein saiddisease status is responsiveness of said patient to treatment with adimer-acting drug.
 33. The method of claim 31 wherein said cancer isselected from the group consisting of breast cancer, ovarian cancer,prostate cancer, and colorectal cancer.
 34. The method of claim 31wherein said one or more ErbB cell surface receptor complexes areselected from the group consisting of Her1-Her1 homodimers, Her2-Her2homodimers, Her1-Her3 receptor dimers, Her2-Her4 receptor dimers,Her1-PI3K complexes, Her2-PI3K complexes, Her3-PI3K complexes, Her1-SHCcomplexes, Her2-SHC complexes, Her3-SHC complexes, Her1-IGF-1R receptordimers, Her2-IGF-1R receptor dimers, Her3-IGF-1R receptor dimers,Her1-PDGFR receptor dimers, Her2-PDGFR receptor dimers, Her3-PDGFRreceptor dimers, p95Her2-Her2 receptor dimers, EGFRvIII-Her1 receptordimers, and EGFRvIII-Her3 receptor dimers.
 35. The method of claim 34wherein said one or more ErbB cell surface receptor complexes areselected from the group consisting of Her1-Her1 homodimers.
 36. Themethod of claim 31 wherein said one or more ErbB cell surface receptorcomplexes each at least one Her2 receptor and at least one of eitherPI3K or SHC.
 37. The method of claim 31 wherein said cancer is selectedfrom the group consisting of breast cancer, ovarian cancer, prostatecancer, and colorectal cancer.
 38. The method of claim 31 wherein saidpatient sample is said fixed tissue sample or said frozen tissue sample.39. A method of determining a cancer status of a patient suffering froma cancer characterized by aberrant expression of one or more ErbB cellsurface receptor complexes, the method comprising the steps of:measuring directly in a patient sample an amount of each of one or moreErbB cell surface receptor complexes; comparing each such amount to itscorresponding amount in a reference sample; correlating differences inthe amounts from the patient sample and the respective correspondingamounts from the reference sample to the disease status the patient; andwherein said one or more ErbB cell surface receptor complexes eachcomprise a Her receptor and an intracellular adaptor molecule.
 40. Themethod of claim 1 wherein said patient sample is a fixed tissue sample,a frozen tissue sample, or circulating epithelial cells.
 41. The methodof claim 40 wherein each of said one or more ErbB cell surface receptorcomplexes are determined by the steps of: providing for each of said oneor more ErbB cell surface receptor complexes a reagent pair comprising acleaving probe having a cleavage-inducing moiety with an effectiveproximity, and one or more binding compounds each having one or moremolecular tags attached thereto by a cleavable linkage, the moleculartags of different binding compounds having different separationcharacteristics; mixing the cleaving probe and the one or more bindingcompounds for each of said one or more ErbB cell surface receptorcomplexes with said patient sample such that the cleaving probe and theone or more binding compounds specifically bind to their respective ErbBcell surface receptor complexes and the cleavable linkages of the one ormore binding compounds are within the effective proximity of thecleavage-inducing moiety so that molecular tags are released; andseparating and identifying the released molecular tags to determine thepresence or absence or the amount of said one or more ErbB cell surfacereceptor complexes in said patient sample.
 42. The method of claim 41wherein said patient sample is said fixed tissue sample or said frozentissue sample.
 43. The method of claim 42 wherein said cancer isselected from the group consisting of breast cancer, ovarian cancer,prostate cancer, and colorectal cancer.
 44. The method according toclaims 39, 40, 41, 42, or 43 wherein said one or more ErbB cell surfacereceptor complexes include one or more complexes selected from the groupconsisting of Her1-PI3K complexes, Her2-PI3K complexes, Her3-PI3Kcomplexes, Her1-SHC complexes, Her2-SHC complexes, Her3-SHC complexes,IGF-1R-PI3K complexes, IGF-1R-SHC complexes, PDGFR-PI3K complexes, andPDGFR-SHC complexes.
 45. The method according to claim 44 wherein saidcancer status is responsiveness of said patient to treatment with adimer-acting drug.